Characterization of Pasteurella multocida associated with pneumonia in bighorn sheep

Pasteurella multocida is a highly diverse group of bacteria recognized as important pathogens. Although P. multocida is not ordinarily associated with disease in Rocky Mountain bighorn sheep (Ovis canadensis canadensis), numerous isolates were cultured in high numbers from free-ranging bighorn sheep in the Hells Canyon area of Idaho, Washington, and Oregon (USA) during the winter of 1995-96. Animals captured in Hells Canyon and held in captivity, and their offspring, also harbored P. multocida. Biochemical utilization tests on 90 isolates identified three subspecies: P. multocida multocida a (n = 54); P. multocida multocida b (n = 13); and P. multocida gallicida (n = 15); and a non-speciated biotype, U6 (n = 8). Genomic DNA digestion with restriction endonuclease Hha I separated the isolates into 62 unique restriction fragment length polymorphism profiles. Capsular type A was predominant (72% of isolates). Only one isolate type, which may have been transmitted from a feral goat, was capsular type D, possessed the structural gene, toxA, for dermonecrotoxin detected by polymerase chain reaction, and produced toxin as determined by monoclonal antibody immunoblot. In conclusion, bighorn sheep in this study carried diverse types of generally non-toxigenic P. multocida associated with epizootic pneumonia.     

An enzyme-linked immunosorbent assay to detect serum IgG to Pasteurella multocida in naturally and experimentally infected rabbits

An enzyme-linked immunosorbent assay (ELISA) was evaluated for efficacy in detecting serum IgG against Pasteurella multocida in both naturally and experimentally infected rabbits. Blood samples and nasal cultures were taken concurrently from 58 rabbits from four conventional rabbitries. Nine rabbits from a pasteurella-free colony served as negative controls. Fifty-six rabbits were ELISA positive. Of these, 46 were P. multocida culture positive, 10 were culture negative. Two rabbits were ELISA negative, culture negative. There were no ELISA negative, culture positive animals. Serotyping by the gel diffusion precipitin test demonstrated that of the 44 typed P. multocida isolates, 57% were serotype 4, 27% were serotype 12 and 16% were serotype 3. In rabbits experimentally infected intranasally with P. multocida, serum IgG against P. multocida began to rise 21 to 33 days after infection and remained elevated until the animals were euthanized 90 days post infection. Two enzyme-linked immunosorbent assays were compared which used potassium thiocyanate extracts of different serotypes of P. multocida as antigen. The results obtained were similar, suggesting the presence of antigens common to both serotypes.     

Colonization of rabbits by Pasteurella multocida: serum IgG responses following intranasal challenge with serologically distinct isolates.

Enzyme-linked immunosorbent assays (ELISAs) and immunoblots were used to measure serum IgG responses in rabbits which were intranasally challenged with Pasteurella multocida. The responses to two serologically distinct isolates (isolate 1, serotype 3:A and isolate 10, serotype 1:D) were compared and then correlated with the ability of the isolates to colonize the nasal passages. Five rabbits were challenged with each isolate (10(5) CFU); nasal washings and sera were collected weekly for 8 weeks. Serum IgG levels were measured by ELISA and immunoblots, using bacterial whole cells and lipopolysaccharides (LPSs) as antigens. The serum IgG response to isolate 1 was evident earlier and was significantly stronger than the response to isolate 10 (P less than 0.025). Immunoblots supported this observation and confirmed that both isolates elicited antibodies which reacted with bacterial protein and LPS antigens, with antibody to protein detectable before antibody to LPS. Results of weekly nasal cultures suggested that the antibody response data could be explained by a difference in the ability of the isolates to colonize the nasal passages: isolate 1 was recovered from four of five rabbits for 8 weeks, whereas isolate 10 was recovered for a maximum of 2 weeks, even when the challenge dose was increased tenfold. The strong response elicited by isolate 1 was therefore probably a result of persistent colonization, whereas the weak response to isolate 10 may have resulted from an inability to persistently colonize the nasal passages. The results of this study demonstrate that isolates of P. multocida elicit antibody responses of differing intensities and vary in their ability to colonize the nasal passages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron microscopic visualization of capsular material of Pasteurella multocida types A and D labeled with polycationic ferritin.

The presence of capsular material on cells of Pasteurella multocida types A and D was determined by transmission electron microscopy after polycationic ferritin labeling. The capsule of agar-grown isolates of P. multocida type A was thick (70 to 90 nm) and regular, whereas that of type D isolates was thinner (20 to 30 nm) and irregular. Such layers were seen on cells from 4- to 6-h broth cultures, but cells from older cultures (12 to 18 h) had very little cell-associated capsular material. Our data indicate that the capsular material of P. multocida types A and D is morphologically different and that capsule production in broth culture is maximal during early log phase.     

Naturally acquired Pasteurella multocida subsp. multocida infection in a closed colony of rabbits: characteristics of isolates

Twelve litters, comprising 41 rabbits aged 35 to 60 days old, in a closed university colony, were monitored for acquisition of nasal Pasteurella multocida subsp. multocida infection. Isolates from 11 infected rabbits were characterized by colonial morphology, capsular type, biotype and antibiotic resistance. Selected isolates were further characterized by somatic antigen typing. Two major strains of P. multocida subsp. multocida were detected in the colony. One strain had mucoid colonies, fermented few carbohydrates and was serotype A:5, whereas, the other strain had smooth iridescent colonies, non-typeable capsular antigen, type 3 somatic antigen and fermented more than twice as many carbohydrates.     

Molecular characterization of Pasteurella multocida isolates from rabbits

Forty isolates of Pasteurella multocida from healthy (17 isolates) and diseased (23 isolates) rabbits were assayed for the presence of plasmids in seeking to determine whether any correlation exists between the presence of plasmids and health status, sensitivity to antimicrobial agents, capsular and somatic type, and the anatomic site of isolation. Six isolates were found harboring plasmids. A similar ladder pattern ranging from 18 to 3 megadalton (Mda) were found in three isolates recovered from diseased rabbits. One band of molecular weight 6.6 Mda was shared by four of five (4/5) isolates from the diseased rabbits. No correlation was found between the presence of the common plasmids and serotype, resistance to antimicrobial agents, and anatomic sites from which the bacteria were cultured. Random amplification polymorphic DNA was applied to subtype all the isolates of P. multocida. Two single primers were tested for their abilities to generate individual fingerprints by using PCR. Primer 1 grouped the isolates into 7 profiles, and primer 2 grouped them into 15. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) results show the presence of a wide heterogeneity within P. multocida isolates. Therefore RAPD-PCR is an efficient technique to detect the DNA polymorphism and could be used to discriminate P. multocida of rabbit isolates together with serologic typing.     

Immunization of rabbits against Pasteurella multocida using a commercial swine vaccine

Elaboration of heat-labile toxin (PMT) is an important virulence factor in some isolates of Pasteurella multocida from rabbits. Previously, we reported that immunization with inactivated PMT (IPMT) stimulated protective immunity to challenges from PMT. To test the hypothesis that immunization with a commercial swine vaccine containing IPMT stimulates similar protective immunity, groups of five rabbits were inoculated twice intramuscularly (i.m.), 10 days apart, with 0.5 ml of sterile saline or a commercial swine P. multocida bacterin-toxoid (BT). In addition, a group was inoculated intranasally with 5 microg of IPMT. Serum and nasal lavage samples were taken on days 0, 7, 14 and 21 after initial immunization and assayed by ELISA for anti-PMT antibody. Serum IgG and nasal lavage IgA were detectable by day 14 in BT and IPMT-immunized rabbits, but not in the saline controls. Groups of similarly inoculated rabbits were then challenged intranasally with 28 microg of PMT 21 days after initial immunization, and necropsied 7 days later, along with control challenged and non-challenged rabbits. Histological lesion severity was graded on a numerical scale. Non-immunized and saline, challenged controls developed more severe pneumonia, pleuritis, nasal turbinate atrophy and testicular atrophy than IPMT and BT-immunized rabbits. The results confirm the hypothesis that immunization with a commercial swine P. multocida BT confers protective immunity in rabbits against challenges from PMT.     

Detection of Escherichia coli cytotoxins by enzyme-linked immunosorbent assays

Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed to detect Escherichia coli cytotoxins. Wells were coated with monoclonal antibodies from hybridomas 13C4 and (or) 11E10, and biotin conjugates of these antibodies were used for detecting verotoxin 1 and Shiga-like toxin II, respectively. Sensitivities were about 100 and 200 cytotoxic doses, respectively. Verotoxin 2 was detected by ELISA with monoclonal antibody 11E10, but at a sensitivity of only about 4000 cytotoxic doses. ELISA results of polymyxin-treated cell extracts from cultures of 67 E. coli strains were in agreement with Vero cell assay as regards the presence and type of toxin.     

Purification and characterization of macrolide 2''-phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics

Hyperimmune and high-titered polyclonal pneumococcal antisera, specific for cross-reactive types within groups, were produced in adult rabbits. Purified capsular polysaccharide was injected intravenously into adult rabbits. One week later, these rabbits were given multiple intravenous injections of formalin-inactivated pneumococci of the cross-reactive type by an established method. Each of the resultant antisera were specific for the cross-reactive type indicating that the previous injection of the polysaccharide had induced epitope-specific tolerance. This method was successful for production of antisera against pneumococcal types 6A, 6B, 9N, 9V, 19F and 19A. Polyclonal rabbit pneumococcal antisera have some advantages over murine monoclonal antibodies for serologic studies and this method should be applicable for producing type-specific antibodies to cross-reactive polysaccharides of clinical interest. Further, this method is simpler and generally produces higher titered monovalent (factor) reagents than absorbed antisera.     

Production of Shiga toxin and a cytolethal distending toxin (CLDT) by serogroups of Shigella spp.

Abstract 56 strains of Shigella including 12 Shigella dysenteriae (serotypes 1, 2, 9, 11 and 12), 23 Shigella flexneri (serotypes 1, 2, 3, 4, 6, var. X and var. Y), 19 Shigella boydii (serotypes 1, 2, 4, 5, 7, 11, 13, 14, 15 and 18), and 2 Shigella sonnei were screened for their ability to produce both classic Shiga toxin and a new heat-labile cytolethal distending toxin (CLDT). Whereas extracellular Shiga toxin was only detectable in filtrates of five S. dysenteriae type 1 strains, CLDT was produced by four strains of S. dysenteriae type 2 and an isolate of S. boydii type 7. No cytotonic enterotoxins similar to Escherichia coli LT were observed in this study. None of the S. flexneri or S. sonnei isolates tested were found to produce extracellular cytotoxic factors. The Shiga toxin produced by the S. dysenteriae type 1 was neutralizable by anti-toxin to verotoxin 1 of E. coli O157 : H7. The Shigella CLDT was neutralizable by antisera prepared to a CLDT-producing E. coli O55 : H4.     

Serologic methods for detection of Pasteurella multocida infections in nasal culture negative rabbits

An agar gel-diffusion test (AGDT) and an enzyme-linked immunosorbent assay (ELISA) were utilized to detect serum antibodies against Pasteurella multocida in naturally infected rabbits derived originally from a Pasteurella-free colony. The antigen used in both assays was purified from a serotype 3 (P-1059) strain of P. multocida. Among 47 serum samples tested 15 (32%) were seropositive; 12 (26%) of which were both AGDT and ELISA-positive, while 3 (6%) were ELISA-positive only. All rabbits examined were normal clinically and negative to repeated nasal cultures, but subsequent cultures at necropsy demonstrated the presence of P. multocida in 11 of the AGDT-positive rabbits and in 14 of the ELISA-positive rabbits. The organism was isolated most frequently from the naso-oropharynx and the tympanic bullae. Serotyping of isolates recovered from the nasopharynx were determined to be serotype 3 or 3,12. Ten seronegative rabbits also were necropsied and none were found harboring P. multocida. These preliminary data indicate that the application of an enzyme-linked immunosorbent assay may prove efficacious in identifying apparently healthy, consistently nasal culture-negative rabbits as subclinical carriers of P. multocida.     

Immunoassay identification ofAspergillus flavus using monoclonal antibodies raised to the whole cell extracts

Five separate monoclonal antibodies were produced against whole cell extracts ofAspergillus flavus and ELISA procedures used to characterise the reactivity of the antibodies to various fungal extracts. All five antibodies were specific to the aflatoxin producing fungi,A, flavus andA. parasiticus, and indicated no cross reactivity with otherAspergillus species, genera of several fungi or with other components which may be found in food samples whereA. flavus may be found.     

Safety and efficacy of a streptomycin dependent live Pasteurella multocida vaccine in rabbits

The safety of and protection provided by a streptomycin dependent live Pasteurella multocida (serotype 12:A) vaccine was evaluated in New Zealand white rabbits. The vaccine strain was isolated from two of twelve rabbits 24 hours after intranasal administration. Streptomycin independent P. multocida isolates were not recovered for 4 weeks after vaccination, indicating a lack of reversion to the wild type. Thirty days after a single intranasal administration of vaccine, eight rabbits were challenged with either P. multocida serotype 3:A or serotype 12:A. Eight non-vaccinated rabbits were challenged in the same manner. Vaccinated rabbits challenged with serotype 12:A had nasal infections for only 2 weeks following challenge. Vaccinated rabbits challenged with serotype 3:A developed chronic nasal infections but were protected from severe disease. Immunoglobulin A or G antibodies against P. multocida were not detected after vaccination in nasal lavages or sera using an enzyme-linked immunosorbent assay. However, both antibodies increased following challenge with either serotype 3:A or serotype 12:A. These studies indicated that the streptomycin dependent pasteurella strain colonized rabbits briefly and was genetically stable in vivo. The results in challenged rabbits suggest that the vaccine provided protection against chronic infection by a homologous pasteurella serotype and protection against severe disease by a heterologous pasteurella serotype.     

Monoclonal antibodies specific for cell culture mycoplasmas

Mycoplasma infection of cell cultures is still a major problem in some laboratories. Although several methods can be used for their detection, identification is normally by serological procedures. As no commercial source for the necessary antibodies is available we have prepared monoclonal antibodies to the five mycoplasma species that account for the majority of cell culture infections. These antibodies have been characterized by the growth inhibition test (GIT), immunofluorescence, and enzyme linked immunosorbent assay (ELISA) and have shown perfect correlation in all tests when compared to conventional antisera raised in rabbits or donkeys. In addition, a monoclonal antibody to Mycoplasma pneumoniae was produced. M. pneumoniae is an infrequent cell culture contaminant but is a human pathogen, and the monoclonal antibody described here could be useful in the clinical diagnosis of M. pneumoniae infection in man.     

Characteristics of Clostridium botulinum Type F Isolated from the Pacific Coast of the United States

Some of the physiological and biochemical characteristics of a type F strain recently isolated from the United States were studied and compared with those of the prototype Langeland type F strain. The recent isolates were nonproteolytic, fermented sucrose and ribose, produced spores of low thermal resistance, produced a protoxin activated by trypsin, and grew and produced toxin at 38 F (3.3 C) from a spore inoculum. The prototype Langeland strain was proteolytic, did not ferment sucrose or ribose, and produced spores of relatively high thermal resistance, and the toxin of 3-day-old cultures was not activated by trypsin. Approximately two to three times the minimal lethal dose (MLD) of type F toxin from either Langeland or nonproteolytic strains was cross-neutralized by 1,000 anti-MLD of type E antitoxin. Antitoxin serums prepared by immunizing rabbits with the toxoid of the nonproteolytic type F isolate neutralized the toxin of the Langeland strain, but did not show cross-neutralization with the toxins of other types of Clostridium botulinum.     

Reactivity of monoclonal islet cell antibodies on normal hyperplastic and neoplastic thyroid C-cells

Summary Thyroid C-cell reactivity to 15 monoclonal antibodies raised against a series of pancreatic islet cells (H[human]ISL, B[bovine]ISL and R[rat]ISL) was evaluated using an indirect immunoperoxidase technique on frozen thyroid sections. Of the monoclonal anti-islet cell antibodies, five reacted specifically with bovine C-cells or human hyperplastic and neoplastic C-cells but not with follicular cells. Two monoclonal antibodies of the bovine series showed strong immunoreactivity with C-cells and only a weakly positive immunostaining of follicular cells. Five monoclonal antibodies reacted with both thyroid C-cells and follicular cells, whereas 3 monoclonal anti-islet cell antibodies did not stain any cell type of the thyroid. In human medullary carcinomas, calcitonin- and somatostatin-producing neoplastic cells were immunoreactive with the same monoclonal antibodies as were normal human C-cells. The protein bands identified by the monoclonal antibodies in human medullary carcinomas had the same molecular weight as those from pancreatic islet extracts. Our study demonstrates the presence of similar differentiation antigens on thyroid C-cells and pancreatic islet cells; this further illustrates common modes of differentiation and specialisation of these embryologically different members of the dispersed neuroendocrine system. The crossreactivity of seven of the monoclonal antibodies investigated with follicular epithelium of the thyroid suggests the existence of common antigenic determinants in different endocrine organs and may partly explain the multiple organ autoimmune response found in patients with polyendocrine diseases.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified ELISA method for detecting Clostridium botulinum type B toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated 80 degrees C/5 min followed by 70 degrees C/2 h before incubation at 15 degrees, 20 degrees or 27 degrees C. Presence of specific toxin was confirmed by mouse bioassay and results were compared with those of the amplified ELISA method. A total of 48 strains, consisting of 38 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 140 slurry samples were tested. Cultures of eight out of nine strains of type B Cl botulinum and 73 of 101 slurry samples containing type B toxin were positive by ELISA; the remaining 28 slurry samples contained type B toxin at levels below or close to the detection limit (20 LD50/ml) of the type B ELISA. No false-positive reactions occurred with Cl. botulinum types A, C, D, E or F, or with the 10 strains of Cl. sporogenes. Toxin produced by one strain of Cl. botulinum type B (NCTC 3807) was not detected by this single monoclonal antibody-based amplified ELISA. With a mixture of two monoclonal antibodies, however, the toxin from NCTC 3807 could be detected without reducing the sensitivity of the ELISA.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified ELISA method for detecting Clostridium botulinum type B toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5–4.5%w/v) and polyphosphate (0.3%w/v) were either unheated or heated 80°C/5 min followed by 70°C/2 h before incubation at 15°, 20° or 27°C. Presence of specific toxin was confirmed by mouse bioassay and results were compared with those of the amplified ELISA method. A total of 48 strains, consisting of 38 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 140 slurry samples were tested. Cultures of eight out of nine strains of type B Cl. botulinum and 73 of 101 slurry samples containing type B toxin were positive by ELISA; the remaining 28 slurry samples contained type B toxin at levels below or close to the detection limit (20 LD₅₀/ml) of the type B ELISA. No falsepositive reactions occurred with Cl. botulinum types A, C, D, E or F, or with the 10 strains of Cl. sporogenes. Toxin produced by one strain of Cl. botulinum type B (NCTC 3807) was not detected by this single monoclonal antibody-based amplified ELISA. With a mixture of two monoclonal antibodies, however, the toxin from NCTC 3807 could be detected without reducing the sensitivity of the ELISA.     

Partial characterization of plasmids from rabbit isolates of Pasteurella multocida.

Plasmids have not been reported for isolates of Pasteurella multocida from rabbits. We assayed 28 isolates of rabbit P. multocida for plasmids and sought to determine whether or not plasmid presence correlated with clinical or pathologic findings, serotype, toxin production, possession of pili, or biochemical characteristics. Fourteen isolates bore a single 1.6 Md (covalently closed circular form in 0.7% agarose gels) plasmid. An additional isolate had two plasmids which migrated as a closely-spaced doublet, centered around 1.6 Md. Eleven isolates appeared to have identical plasmids, according to Hae III and Hinf I digests. The apparent linear size of this common plasmid in 2% agarose gels was 2.1 Md, as calculated from the sums of the sizes of Hae III or Hinf I digestion fragments. Linearization of the common plasmid with Msp I produced an apparent size of 2.5 Md in 0.7% agarose gels. No correlations between presence of the common plasmid and somatic serotype, toxigenicity, presence of pili, antimicrobial resistance, selected biochemical characteristics, anatomic site from which the bacteria were cultured, or disease status of the host were found.