Naturally acquired Pasteurella multocida infection in rabbits: immunological aspects

Natural infection with P. multocida in New Zealand White rabbits was followed in 2 to 3 weeks by development of immunoglobulin (Ig)M and IgG serum antibodies. The IgM response peaked and returned to lower levels within several weeks after infection, whereas the IgG response progressively increased and remained elevated.     

Transmission of Pasteurella multocida in rabbits

Contact and airborne transmission of P. multocida in rabbits was evaluated in an artificially controlled environment. Transmission by contact occurred more readily from rabbits with acute infections than from rabbits with chronic infections. However, airborne transmission to rabbits in adjacent cages did not occur.     

Experimental respiratory infection with Pasteurella multocida and Bordetella bronchiseptica in rabbits.

Eight-to-10-wk-old offspring of a colony of specific pathogen free [Eda:(NZW x FG)F1BR] rabbits were exposed to cultures of Pasteurella multocida and Bordetella bronchiseptica. Two groups of 9 animals each were exposed to cultures of either species of bacteria intranasally and killed 2, 7, 14, and 21 da postinoculation. Five of 9 rabbits in each group developed a mucopurulent nasal discharge 4-7 da postinoculation. The remaining 4 rabbits in each group failed to develop clinical signs. The gross and microscopic lesions did not differ in character or distribution among the inoculated rabbits. The infection was characterized by an acute upper respiratory syndrome accompanied by a mild bronchopneumonia.     

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.     

A dot-immunobinding assay for the laboratory diagnosis of tuberculous meningitis and its comparison with enzyme-linked immunosorbent assay

V.V. RADHAKRISHNAN AND A. MATHAI. 1991. In an attempt to establish an alternative to standard bacteriological methods in the laboratory diagnosis of tuberculous meningitis (TBM), a simple dot-immunobinding assay (Dot-Iba) was standardized to detect Mycobacterium tuberculosis antigen 5 and antimycobacterial antibody in cerebrospinal fluid (CSF) specimens of patients with TBM. Sensitivity and specificity of Dot-Iba was compared with conventional enzyme-linked immunosorbent assay (ELISA) and standard bacteriological techniques. The Dot-Iba showed excellent correlation with indirect ELISA for the detection of antimycobacterial antibody in CSF and showed 60% sensitivity and 100% specificity in culture-negative patients with TBM. However Dot-Iba was less sensitive for the detection of antigen 5 in CSFs and showed false negative results (60%) in culture-positive patients with TBM.     

Heat-labile toxin-producing isolates of Pasteurella multocida from rabbits.

Five of one hundred forty seven isolates of Pasteurella multocida from rabbits were found to produce heat-labile toxin. Each isolate was assayed for the ability of potassium thiocyanate (KSCN) extracts to cause dermonecrosis in guinea pig skin, ability of bacteria or filtrates to cause cytotoxicity in cell cultures, and reactivity with monoclonal antibodies to heat-labile P. multocida toxin. Five capsular type D isolates produced dermonecrosis and reacted with monoclonal antibodies to toxin. Filtrates of all five of these isolates were cytotoxic for cell cultures. Potassium thiocyanate extracts of all five isolates caused pleuritis and pneumonia in rabbits after intranasal inoculation. Turbinate atrophy was seen in 5 of 19 rabbits inoculated intranasally with toxic extracts. Heat-labile toxin was not produced by 109 capsular type A isolates or 19 nontypable isolates.     

A dot-immunobinding assay for the laboratory diagnosis of tuberculous meningitis and its comparison with enzyme-linked immunosorbent assay.

In an attempt to establish an alternative to standard bacteriological methods in the laboratory diagnosis of tuberculous meningitis (TBM), a simple dot-immunobinding assay (Dot-Iba) was standardized to detect Mycobacterium tuberculosis antigen 5 and antimycobacterial antibody in cerebrospinal fluid (CSF) specimens of patients with TBM. Sensitivity and specificity of Dot-Iba was compared with conventional enzyme-linked immunosorbent assay (ELISA) and standard bacteriological techniques. The Dot-Iba showed excellent correlation with indirect ELISA for the detection of antimycobacterial antibody in CSF and showed 60% sensitivity and 100% specificity in culture-negative patients with TBM. However Dot-Iba was less sensitive for the detection of antigen 5 in CSFs and showed false negative results (60%) in culture-positive patients with TBM.     

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.     

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.     

Pasteurella multocida infection in man; a review of the literature

Pasteurella multocida has been isolated from infected lesions of nearly all regions of the body. Cat and dog bite wounds are frequently infected by this organism. The severity of the lesion varies from trivial sepsis to a severe wound abscess with sloughing and septicaemia. Bone involvement is frequent. Despite the apparent sensitivity of the organism to a wide range of antibiotics, chemotherapy may be of little avail. Frequently, extensive débridement and skin grafting is required. In the respiratory tract, the clinical picture extends over bronchitis, bronchiectasis, pneumonia, empyema, otitis media, mastoiditis and sinusitis. In these latter infections a history of contact with animals may not be elicited. Examination of the patient's serum may not be helpful as antibody titers are often low, even in the presence of severe acute infection.     

A dot-immunobinding assay for monoclonal and other antibodies

A procedure for the assay of antibodies in sera based on the application of the antigen as a spot to nitrocellulose filters is described. The method has the merit of being simpler in operation and more sensitive than comparable existing procedures. Applications for screening supernatants of hybridomas making monoclonal antibodies, and the use of such antibodies in the determination of the tissue distribution of the corresponding antigens, are described. An application for the screening of human pathological sera for multiple antibodies in one operation is also described.     

Ultrastructural characteristics of the external surfaces of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits

The surface structures of the cells of Pasteurella pneumotropica from mice and Pasteurella multocida from rabbits were examined by transmission electron microscopy after ruthenium red staining and polycationic ferritin labelling. P. pneumotropica strains ATCC 35149 and K 79114 had slight extracellular fibrous materials associated with cell walls with ruthenium red staining. Ferritin labelling method revealed thick strands or sparsely ferritin-labelled materials on the cell surface of the strains. P. multocida strains Pm-78 and P-2440 had ferritin-labelled capsules surrounded with the cell wall. Strain Pm-78, which was serotyped as A:12, had a thick capsule, whereas serotype -:3 strain P-2440 had a thin and irregular capsule.     

A minimal medium for growth of Pasteurella multocida

Abstract We developed a minimal medium supporting the growth of both toxigenic and nontoxigenic strains of Pasteurella multocida to optical densities of > 0.5 (600 nm ). P. multocida P1059 (ATCC 15742), one of a number of strains which can cause fowl cholera, was used as the model strain in this study. The medium was composed of 17 ingredients including cysteine, glutamic acid, leucine, methionine, inorganic salts, nicotinamide, pantothenate, thiamine, and an energy source. Leucine was not required for growth but was stimulatory, and thiamine could be replaced by adenine. An additional 46 strains of P. multocida were tested, and 40 out of 46 (87%) strains grew as well as strain P1059 through a minimum of 10 serial transfers. P. multocida toxin (PMT) was produced when cells of a known toxigenic strain (P4261) were cultivated in the minimal medium. No growth of Pasteurella haemolytica or Pasteurella trehalosi strains was observed in this minimal medium.     

Development of a 23S rRNA-based PCR assay for the identification of Pasteurella multocida

AIMS: The aim of this work was to develop a rapid diagnostic test for Pasteurella multocida. METHODS AND RESULTS: A polymerase chain reaction (PCR) assay using primers derived from the 23S rRNA gene sequence of Past. multocida was developed. The PCR assay correctly identified all 144 isolates of Past. multocida tested, including type strains of the three subspecies as well as the reference strains for the Heddleston and Carter typing schemes. Of 20 closely related bacteria from the family Pasteurellaceae tested, only the type strains of Past. canis biovar 2 and Past. avium biovar 2 were positive. These two bacteria, formerly known as Bisgaard Taxon 13, are the closest phylogenetic relatives of Past. multocida based on 16S ribosomal rRNA. All phylogenetically unrelated avian and porcine organisms tested were negative. CONCLUSION: This PCR enables rapid identification of Past. multocida colonies from avian or porcine origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Veterinary diagnostic laboratories can use this PCR to rapidly and accurately diagnose fowl cholera and porcine pasteurellosis.     

Immunopotentiation of outer membrane protein through anti-idiotype Pasteurella multocida vaccine in rabbits

Pasteurella multocida was isolated from cattle affected with haemorrhagic septicaemia and characterized on the basis of morphological, cultural and biochemical tests. Bacterial outer membrane proteins (OMPs) were extracted with 1% Sarkosyl method. P. multocida anti-idiotype vaccine prepared from OMPs (21.3 mg per 100 ml), was evaluated and compared with bacterin supplemented with 10% OMPs and plain alum-adsorbed bacterin in rabbit models. It was observed that OMPs-anti-idiotype vaccine induced high levels of antibody titres (geomean titres -GMT) detected using indirect haemagglutination (IHA) test. The OMPs anti-idiotype antibody titres of 168.9 GMT were obtained to 42.2 GMT in OMPs supplemented bacterin on 21 days post vaccination, while the plain bacterin had the least titre of 27.9 GMT. The OMPs-anti-idiotype vaccine provoked better immunogenic response in terms of highest GMT titres and long lasting effect in rabbits and 100% protection against the challenge with homologous strain of P. multocida,while 88% protection was obtained in rabbits, given OMPs supplemented bacterin.