Richard B. Rimler

Capsular hyaluronic acid-mediated adhesion of Pasteurella multocida to turkey air sac macrophages.

Serogroup A strains of Pasteurella multocida, the major cause of fowl cholera, are resistant to phagocytosis in nonimmunized birds. Adherence studies with a capsulated strain of P. multocida (serotype A:3) and turkey air sac macrophages in culture showed that the bacteria were capable of adhering in large numbers to the macrophages but were not internalized. A noncapsulated variant of the bacteria (serotype -:3) showed little or no adherence and was not internalized. These data indicated that the adhesive properties were caused by the presence of a capsule on the bacteria. The role of capsular hyaluronic acid in adherence to macrophages was investigated. Depolymerization of the bacterial capsule with hyaluronidase increased phagocytosis by macrophage cultures, and addition of hyaluronic acid to the macrophages inhibited bacterial adherence. Additionally, exposure of macrophages to chondroitin sulfate B, an anionic polysaccharide similar to hyaluronic acid, did not affect the adhesive properties and resistance to phagocytosis of capsulated organisms. Treatment of macrophages with sodium metaperiodate or trypsin suppressed bacterial binding. Collectively, these data indicate that P. multocida adhesion to air sac macrophages, but not internalization, is mediated by capsular hyaluronic acid and suggest that recognition of this bacterial polysaccharide is a result of a specific glycoprotein receptor.

Purification of a cross-protective antigen from Pasteurella multocida grown in vitro and in vivo.

A peptone-based medium was formulated to grow Pasteurella multocida in vitro, which expressed an antigen that induces cross protection in turkeys against different serotypes. Vaccines of various chromatographic fractions obtained from P. multocida grown in the medium induced active immune cross protection in turkeys, and sera from these turkeys passively cross protected naïve poults. An antigen of approximately 39 kD molecular size was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroelution from hydroxyapatite chromatographic fractions of both in vivo- and in vitro-grown P. multocida. The purified antigen from either source induced active immune cross protection but no passive protection in one of two experiments. Increasing the dose of vaccine resulted in both active and passive immune cross protection in the second experiment.

Turbinate Osteoporosis in Pigs Following Intranasal Inoculation of Purified Pasteurella Toxin: Histomorphometric and Ultrastructural Studies

Turbinate osteoporosis, induced by intranasal inoculation of purified toxin isolated from serotype D Pasteurella multocida, was investigated in 3- to 5-week-old, caesarean-derived, colostrum-deprived, isolation-reared pigs. Marked bilateral reduction in relative volume of trabecular bone occurred in osseous cores of turbinates of toxin-treated pigs relative to control pigs on post-inoculation day (p.i.d.) 3, 6, 9, 12, and 15. The fractional resorptive surface along turbinate bone was greater in toxin-treated pigs when compared to controls on p.i.d. 3 and 6. A significant decrease in resorptive surface occurred over time in toxin-treated pigs, whereas the fractional resorptive surface was constant over time in control pigs. Osteoclasts in medullary spaces separating bony trabeculae of turbinates were abundant in toxin-treated pigs and scant in controls on p.i.d. 3, 6, and 9. Degeneration and necrosis of bone forming cells, principally osteoblasts, were progressively more extensive with time and were associated with decreased mineralization and reduced thickness of osteoid and woven bone matrix. Osteoclasts along resorptive surfaces of turbinate bone in toxin-treated pigs had more abundant, more highly vacuolated cytoplasm, a more prominent microvillous border, and a greater number of nuclei per cell than osteoclasts from control pigs on p.i.d. 3 and 6. We conclude that this Pasteurella toxin stimulates osteoclastic osteolysis and inhibits osteogenesis in turbinates by causing degeneration and death of osteoblasts.

Effects of Pasteurella multocida Toxin on Porcine Bone Marrow Cell Differentiation into Osteoclasts and Osteoblasts

The effect of Pasteurella multocida toxin (PMT) on porcine osteoclast and osteoblast differentiation was studied using in vitro cell culture systems. When grown in the presence of Vitamin D3, isolated porcine bone marrow cells formed multinucleated cells with features characteristic of osteoclasts. Exposure of bone marrow cells to Vitamin D3 and PMT during growth resulted in formation of increased numbers and earlier appearance of osteoclasts compared to controls. Ultrafiltered medium from PMT-treated cells likewise increased osteoclast numbers, suggesting that a soluble mediator may be involved in the action of PMT. When cell cultures were treated with fluorescein-labeled PMT, fluorescence was found within the cytoplasm of small, round cells that did not resemble either osteoclasts or osteoclastic precursor cells. Cultures of porcine bone marrow cells exposed to dexamethasone, ascorbic acid, and β-glycerophosphate developed into osteoblastic cells that formed multilayered, mineralized nodules. Exposure of osteoblastic cultures to low concentrations of PMT resulted in retarded cell growth, formation of decreased numbers of nodules, and minimal to no mineralization in the nodules; higher concentrations of PMT resulted in increased cellular debris and poor growth of cells, with no nodule formation. These findings suggest that PMT may induce turbinate atrophy in pigs by increasing osteoclast numbers and inhibiting osteoblastic bone formation. The effect of PMT on osteoclastic differentiation and growth may not be due to a direct effect on preosteoclastic cells, but rather due to alterations in the soluble mediator secretion by marrow stromal cells.

A Toxin from Pasteurella multocida Type D Causes Acute Hepatic Necrosis in Pigs

Toxins of Pusteurellu multocidu type D, a causal bacterium of porcine atrophic rhinitis, chiefly affect developing cartilage and bone. When sprayed onto nasal mucosae or injected subcutaneously, they cause lesions of nasal turbinates within 48 h r . * ~ ~ Systemic effects of pasteurella toxins have not been clearly shown but are suggested by two experimental studies in pigs: (1) toxin given by aerosol caused hepatocyte damage in six of ten pigs-lesions varied from focal vacuolar degeneration to diffuse necrosis and hemorrhage;2 (2) toxin given intraperitoneally caused liver lesions which were defined only as “degenerative change.”8 Since clinical studies on atrophic rhinitis hint that the liver may be affected in natural disease, we wished to know if the pig developed liver necrosis when given toxin experimentally. Six piglets (specific pathogen-free, 3 days old) were given 0.1 pg (0.1 ml) toxin subcutaneously; two controls (pigs 1, 2) were given diluent subcutaneously. Two pigs were to be necropsied at 2 hr (pigs 3, 4), 24 hr (pigs 5, 6), and 48 hr (pigs 7, 8) after toxin (Table 1). Toxin, purified from noncapsulated Pusteurellu multocidu strain P-4533,4J was suspended in buffer (0.05 M Tris, 0.1 M NaC1,0.002 M EDTA, 0.5% bovine serum albumin, pH 8.3) at a protein concentration of 1 pg/ml; the lethal dose for swine is between 0.1 and 0.8 pg/kg given intradermally.“ From all pigs, slices of liver, kidney, lung, adrenal, stomach, jejunum, thymus, spleen, mesenteric lymph node, and rib were fixed in 10% formalin and processed by standard histologic techniques. After embedment in paraffin blocks, sections (6 pm) were cut and stained with hematoxylin and eosin (HE), Giemsa, oil red 0, and alcian blue-periodic acid-Schiff (with and without diastase) techniques. Livers of normal control piglets had no pathologic changes; hepatocytes contained large amounts of glycogen but little lipid. Livers sampled at 2 hr after toxin (Fig. 1) resembled controls except for three changes, all of which were slight: (1) loss of glycogen and increased density of cytoplasm of hepatocytes surrounding central veins; (2) dilation of the hepatic venous system; and (3) infiltrates of small numbers of neutrophilic leukocytes in surrounding sinusoids. Pathologic changes in livers at 24 hr after toxin were striking and severe (Fig. 2). They included: (1) marked intrahepatic edema; ( 2 ) diffuse swelling and enlargement of Kupffer cells; (3) diffuse endothelial swelling and necrosis with dilation of sinusoids; (4) diffise degeneration of hepatocytes; and (5) random foci of necrosis, which were especially prominent at the surface of the liver. Foci of necrosis were characterized by detachment of necrotic hepatocytes, insudation of plasma proteins, and microcavitation (Fig. 3). Pig 7 died 33 hr after toxin, and pig 8 was killed 36 hr after toxin; lesions did not differ from those in 24-hr samples. Degenerate hepatocytes throughout the livers of piglets sampled at 24 and 36 hr after toxin had marked depletion of glycogen, vacuolar degeneration, and increased basophilia of cytoplasm. Small vacuoles in the cytoplasm of hepatocytes were lucent and did not stain for fat or glycoproteins. In contrast, larger vacuoles contained inspissated protein which stained with the periodic acid-Schiff (PAS) technique. Some appeared as hyalin lakes that filled the cytoplasm (Fig. 4). Endothelial degeneration and necrosis in livers at 24 hr after toxin was diffuse but was most severe in terminal sinusoids and central veins. Neutrophils were prominent in these areas. Perivascular eosinophilic precipitates of protein were around most central veins. Lesions in other organs were more variable than the hepatic lesions. No significant changes were seen 2 hr after toxin. In 24and 36-hr samples, in addition to marked generalized hyperemia, histologic lesions included: (1) small intestine: necrosis of capillaries of the tips of villi with many degenerate rounded enterocytes free in the gut lumen close to the villous tips (Fig. 5); (2) kidney: acute cell swelling and necrosis of segments of the proximal convoluted tubules in developing nephrons (Fig. 6), especially adjacent to developing glomeruli at the surface of the kidney; (3) lungs: slight edema of the alveolar wall with dilation of capillaries, neutrophilic leukocytes within and external to blood vessels, dilatation of lymphatics, and endothelial damage; (4) bone: necrosis of capillaries and fibrin deposition at the junction of cartilage and bone at the growth plate; and ( 5 ) adrenal: focal necrosis in the cortex of two of four glands. No significant lesions were seen in pancreas, thymus, lymph node, or spleen. Studies on blood collected just before necropsy did not reveal marked changes in leukocytes or plasma proteins (Table 1). Complement levels were not significantly reduced. There were consistent elevations in serum sorbitol dehydrogenase associated with liver injury. These studies show that pasteurella toxin causes hepatic’ necrosis associated with widespread vascular injury and vacuolar degeneration of hepatocytes. The mechanism of toxin action on hepatocytes appears to be associated with, but

Use of rats to compare atrophic rhinitis vaccines for protection against effects of heat-labile protein toxin produced by Pasteurella multocida serogroup D

Four bacterin-toxoid and three bacterin commercial vaccines against atrophic rhinitis were tested in rats for their capacity to immunize against the lethal and systemic effects of purified heat-labile protein toxin (D-toxin) produced by Pasteurella multocida serogroup D. Only one bacterin-toxoid vaccine stimulated sufficient immunity to prevent the death of all rats challenged with D-toxin. None of the vaccines prevented weight loss, leukocytosis or increases in serum complement titers in rats challenged with D-toxin. Rats provide an inexpensive animal model for testing the capacity of vaccines to generate antitoxic immunity against the lethal and systemic effects of D-toxin.

DNA Fingerprinting of Riemerella anatipestifer

Seventeen restriction endonucleases were evaluated for use in DNA fingerprinting of Riemerella anatipestifer. Digestion of chromosomal DNA with either HinfI or DdeI restriction endonuclease, followed by submarine electrophoresis in agarose and staining with ethidium bromide, resulted in DNA fingerprint profiles that could be easily resolved. HinfI produced readable fingerprint patterns in the 2.7-20-kb range and was used to distinguish DNA fingerprint profiles among 89 strains of R. anatipestifer representing isolates from various avian species in the United States, the United Kingdom, Australia, Canada, Germany, and Israel. A total of 52 distinct DNA fingerprint profiles were found.

Passive immune cross-protection in mice produced by rabbit antisera against different serotypes of Pasteurella multocida

Haemorrhagic septicaemia (HS) and fowl cholera (FC) are specific diseases caused by certain serotypes of Pasteurella multocida. Strains that usually cause HS in cattle and water buffalo do not produce FC in avian species, and strains that cause FC do not produce HS in cattle and water buffalo. A variety of P. multocida serotypes, including unusual strains which can cause HS in wild ruminants, were evaluated in passive immune protection studies to determine the immunological relationship between strains associated with HS and FC. Various degrees of cross-protection were seen among the strains. Antiserum against a serotype B:3,4 strain protected against strains capable of causing HS (serotypes B:1, B:2, B:3,4, B:4 and E:2) and FC (serotypes A:1, A:3 and A:5). Antiserum against an FC strain (serotype A:5) similarly protected against strains capable of causing HS and FC. Antigenic analyses indicated that cross-protection was not necessarily induced by serotype-specific capsular (beta) or somatic (gamma) antigens or major porin protein. SDS-PAGE and immunoblots of whole cell lysates of the different HS and FC strains showed many protein-staining bands with similar mobilities and antigenic activity. These cross-reactive antigenic bands occurred in the 20- to 120-kDa range. Adsorption of antiserum with a heterologous serotype removed its reactivity with most of these bands, as well as its ability to cross-protect.

Influence of chondroitinase on indirect hemagglutination titers and phagocytosis of Pasteurella multocida serogroups A, D and F

Capsules of Pasteurella multocida serogroups A, D and F contain mucopolysaccharides which block antigenic determinants and prevent phagocytosis. In this study, capsules of serogroup A, D and F strains of P. multocida were depolymerized by enzyme treatment. Capsule depolymerization of serogroup D and F strains with chondroitinase increased indirect hemagglutination (IHA) test titers and enhanced phagocytosis by swine neutrophils. Capsule depolymerization of serogroup A strains with hyaluronidase increased IHA titers, but depolymerization with chondroitinase did not. When serogroup A strains were treated with a combination of chondroitinase and hyaluronidase, IHA test titers were lower than titers of the same strains treated with hyaluronidase alone. Combined enzyme treatment of serogroup D strains resulted in IHA test titers similar to those of chondroitinase treatment alone.

Immunity induced in rats vaccinated with toxoid prepared from heat-labile toxin produced by Pasteurella multocida serogroup D.

Rats were vaccinated with a toxoid (D-toxoid) prepared from purified heat-labile toxin (D-toxin) produced by Pasteurella multocida serogroup D. Vaccination of rats with D-toxoid prevented death and other effects of D-toxin (hepatic necrosis, development of elevated leukocyte counts, lymphopenia, neutrophilia, and elevated complement titers) that occurred in phosphate buffered saline (PBS)-vaccinated control rats.