Comparison of Freund's and Ribi adjuvants for inducing antibodies to the synthetic antigen (TG)-AL in rabbits.

Antibody responses and health parameters were compared in rabbits immunized with a synthetic polypeptide antigen, [L-Tyr,L-Glu,DL-Ala]-poly-L-lysine ((TG)-AL), in Freund's (FA) or Ribi (RA) adjuvants. Rabbits, 12 weeks old, of both sexes, were inoculated with 0.5 ml divided between two intramuscular (i.m.) sites. Eight received FA and antigen (50 micrograms); eight RA and antigen, eight PBS and antigen; four FA and PBS; four RA and PBS, and four PBS. Identical booster inoculations were made 21 days later, except that incomplete FA was substituted for complete FA. Rabbits were monitored until euthanasia and necropsy 7 weeks after the primary inoculation. Sera, obtained weekly, were analyzed for immunoglobulins using an enzyme immunoassay. Only rabbits given antigen with adjuvant produced high titered antibodies. Mean optical density values for immunoglobulin (Ig)M were greater the week after the booster in the group given FA. IgG values were similar for both adjuvant/antigen groups the week after the booster, but thereafter decreased in rabbits given RA. Antisera from rabbits given antigen with FA had greater avidity for the antigen than that from rabbits given antigen with RA, however, the difference was not significant (p greater than 0.05). Rabbits inoculated with FA and antigen had high serum creatinine kinase levels the day after inoculation, showed evidence of discomfort, and extensive granulomatous inflammation at the inoculation sites. Lesions were minimal to mild in rabbits given antigen with RA and PBS with either adjuvant. While RA did not result in adverse side effects, the IgG response to (TG)-AL with RA was transient compared to FA.

Polypeptides associated with Pasteurella multocida infection in rabbits.

Polypeptides from whole cell preparations of Pasteurella multocida serotypes A:12 and A:3 were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Antigens were detected by immunoblot analysis, using sera from 3 groups of rabbits. Sera were obtained from rabbits inoculated intranasally with P multocida serotype A:12 or A:3, from rabbits maintained in a rabbitry with enzootic P multocida A:12 infection, and from rabbits maintained in a rabbitry with enzootic P multocida A:3 infection. Immunoblot analyses of pre- and postinoculation sera from experimentally infected rabbits, using serotype A:12 antigen, revealed 3 polypeptides with approximate molecular mass of 28, 30, and 37 kDa that consistently detected antibodies after P multocida-induced infection. Sera from rabbits naturally infected with either serotype, tested against serotype A:12 and A:3 antigens, detected the same polypeptides in both serotypes. Thus, immunologic reactivity to these polypeptides may be useful for serologic detection of P multocida infection.

Atrophic rhinitis in New Zealand white rabbits infected with Pasteurella multocida.

Atrophic rhinitis was detected in New Zealand White rabbits when upper respiratory tract disease was evaluated during a vaccine field trial for the prevention of pasteurellosis. Of 52 adult rabbits euthanatized and necropsied, 26 (50%) had evidence of turbinate atrophy. Atrophy was detected in 77% of rabbits with Pasteurella multocida infection only, 71% of rabbits with concurrent P multocida and Bordetella bronchiseptica infections, and 6% of rabbits with B bronchiseptica infection only. Grossly, turbinate atrophy was characterized by a mild to severe loss or diminution in the maxilloturbinates. Histologically, turbinate bones were small and irregular in thickness and had numerous osteoclasts and osteoblasts. A neutrophilic exudate filled the nasal passages, and infiltrates of neutrophils and lymphocytes were detected in the mucosa and submucosa of the nasal turbinates. Rhinitis was significantly (P less than 0.001) associated with turbinate atrophy. Isolates of P multocida from rabbits with turbinate atrophy were serotype A:12.

Toxin production by Pasteurella multocida isolated from rabbits with atrophic rhinitis.

Naturally acquired turbinate atrophy in rabbits was associated with Pasteurella multocida infection. Several in vitro and in vivo studies were conducted to document toxin production from P multocida isolates and to determine the relation of toxin to atrophic rhinitis in rabbits. Ten isolates of P multocida serotype A:12 were obtained from adult New Zealand White rabbits with noninduced atrophic rhinitis. Specific-pathogen-free rabbits inoculated intranasally with isolates of P multocida developed rhinitis and turbinate atrophy. However, inoculation with filtrates of the same bacteria failed to induce turbinate atrophy. Cytotoxicity was observed in assays, using bovine embryonic turbinate cell cultures with extracts of P multocida, but not in agar overlay cytotoxicity assays, using bovine embryonic turbinate, bovine embryonic lung, or Vero cell cultures, or in a sandwich ELISA, using monoclonal antibodies to purified P multocida toxin. Thus, turbinate atrophy was experimentally reproduced in rabbits with isolates of P multocida, but toxin was only detected in vitro by cell culture assay of P multocida extracts.

Guineapig inclusion conjunctivitis (GPIC) in a commercial colony.

Serological findings in a commercial colony of Hartley guineapigs revealed that about 70% had antibodies to Chlamydia psittaci as detected by the microimmunofluorescence method. Conjunctivitis was evident in 14% of 86 guineapigs examined. Chlamydial antigen was detected in conjunctival scrapings by a direct immunofluorescence test using Chlamydia-specific monoclonal antibody; however, C. psittaci was not demonstrated by other methods.

Cerebral larva migrans caused by Baylisascaris sp in pet rabbits.

Cerebral larva migrans was diagnosed histologically in 4 pet rabbits that developed progressive neurologic disease. Larvae of Baylisascaris sp were isolated from brain tissues in 2 rabbits. The clinical syndrome of progressive torticollis and ataxia manifested by these rabbits is commonly associated with otitis and labyrinthitis attributable to bacterial infection; however, the middle ears were normal on radiographic and postmortem examinations. The severe encephalopathy that developed in these rabbits was indicative that just a few Baylisascaris larvae may cause extensive brain injury. During the summer, all of the affected rabbits were maintained outdoors in suburban areas, where raccoons, the final host of B procyonis, are commonly observed. Raccoon feces containing B procyonis eggs constitute a health risk for rabbits, as well as for human beings.

Respiratory diseases of rabbits.

Respiratory diseases are second only to gastroenteric diseases in importance in rabbits. Pasteurellosis is the primary respiratory disease affecting domestic rabbits, but other bacteria (e.g., Bordetella broniseptica and Staphylococcus spp) are significant opportunistic pathogens. The primary manifestations are upper respiratory disease (e.g., rhinitis, sinusitis, conjunctivitis, and dacryocystitis). Various antimicrobials are effective for treatment.

Characterizion of Mycoplasma pulmonis variants isolated from rabbits. II. Basis for differentiation of antigenic subtypes.

The antigen composition of Mycoplasma pulmonis variants was studied by complement-fixation, agar-gel diffusion, and growth-inhibition tests. Two classes of complement-fixing antigens were demonstrated for M. pulmonis strains 47 and 63: (i) cross-related, heat-labile, water-soluble antigens, and (ii) high-titered, subtype-specific, heat-stable, water-soluble antigens. Lipid antigens prepared by organic solvent fractionation were low-titered antigens and showed little specificity. With the aid of agar-gel double-diffusion plates, the subtype-specific antigens were found to be precipitated by trichloroacetic acid and to be stable to periodate, but they were inactivated by pronase. Pronase-stable, periodate-labile precipitating antigens were observed as common components between the two variants. Antisera prepared with boiled antigens were found to be serologically active on gel diffusion but lacked neutralizing ability in growth-inhibition tests. Each of three strains of M. pulmonis (47, 63, ATCC 14267) could be identified as a variant because each strain possessed immunologically distinct heat-stable subtype-specific antigen(s).

Characterization of Mycoplasma pulmonis variants isolated from rabbits. I. Identification and properties of isolates.

Mycoplasma showing at least two colony types were isolated from the nares and oropharynx of New Zealand white rabbits. Two strains were purified by single-colony passages and characterized. Morphology by phase-contrast and electron microscopy was typical of Mycoplasmataceae. Both grew anaerobically as well as aerobically, caused hemolysis of guinea pig, sheep, and horse red blood cells, and fermented glucose. These characteristics are shared by members of the species M. pulmonis, commonly isolated from the respiratory tracts of laboratory rats and mice. By use of the growth-inhibition test and agar-gel double-diffusion tests, the two strains were found to be serologically related to each other and to M. pulmonis ATCC 14267 but not to other representative Mycoplasma species from man and animals.

Detection of Antibodies to Pasteurella multocida by capture enzyme immunoassay using a monoclonal antibody against P37 antigen.

As infection with Pasteurella multocida is common in rabbits, an enzyme immunoassay (EIA) was developed for its detection. A murine immunoglobulin G monoclonal antibody was used to capture a 37-kDa polypeptide of P. multocida serotype A:12 in an EIA to detect antibodies to P. multocida. The 37-kDa antigen was selected since it was previously shown to be a major immunogen during P. multocida infection in rabbits. The sensitivity of the P37 EIA, determined with sera from 56 rabbits infected with P. multocida, was 98%. Specificity, evaluated with sera from 62 rabbits from colonies free of P. multocida, was 92%. Titration curves of sera from rabbits immunized with P. multocida serotype A:3 or A:12 coincided, indicating that the P37 EIA was equally efficient in detecting antibodies to the two major serotypes of the organism. Comparison of the P37 EIA with the current serodiagnostic test, a bacterial lysate EIA, revealed relatively good correlation (r = 0.68). However, specificity was greatly improved, as 34% of uninfected rabbits were falsely positive by the lysate EIA whereas only 3% of uninfected rabbits were falsely positive by the P37 EIA. The coefficient of variation for same-day tests was 10%, and that for interday tests was 15%, indicating good reproducibility. The greater sensitivity and specificity of the P37 EIA should significantly enhance diagnostic capability to identify rabbits infected with P. multocida.

Toxoplasma gondii infection of cats in Beirut, Lebanon.

The prevalence of serum antibodies to Toxoplasma gondii was determined by the indirect fluorescent antibody (IFA) test in cats of Beirut, Lebanon from September 1980 to July 1983 (35 months). Testing revealed that 253/324 (78.1%) cats had antibodies in the IFA test. While the prevalence of Toxoplasma antibodies in stray cats was considerably greater than in owned cats at the beginning of the study, this difference diminished over time, and prevalence was similar by the end of the study. Faecal shedding of Toxoplasma-like oocysts was found in 31/313 (9.9%) of cats, while 38/313 (12.1%) shed other Isospora spp. oocysts. The occurrence of dual infections was statistically significant.

The effect of combined rotavirus and Escherichia coli infections in rabbits.

In rabbits, experimentally induced rotavirus infection results in soft feces only; thus it is unlikely that it is the sole cause of the severe, often fatal diarrhea of weanling rabbits with which it is associated. To determine whether rotavirus acts synergistically with another pathogen, New Zealand White rabbits (10 to 38 weeks old) were inoculated with rotavirus (L:ALA:84) and/or Escherichia coli 015:H-(RDEC-1) via orogastric tube. A single dose of high-titer (10(6) fluorescent focus-forming units) rotavirus was used, whereas E. coli was administered in various doses (10(2) to 10(9) CFU) to determine the titer of E. coli that induced only mild diarrhea but, when combined with rotavirus, resulted in diarrheal disease. Doses of E. coli > 10(6) CFU resulted in infection in almost all rabbits 10 to 16 weeks old, as detected by fecal shedding, regardless of whether rotavirus was inoculated simultaneously. However, inoculation of > 10(6) CFU of E. coli, in conjunction with rotavirus, resulted in increased morbidity and mortality due to diarrheal disease compared with E. coli alone. Inoculation of rabbits 28 to 38 weeks old with similar doses of rotavirus and E. coli caused infection but failed to induce diarrhea, indicating that older rabbits were more resistant to the pathogenic effects of these two agents. A synergistic effect between rotavirus and E. coli occurred, causing more severe diarrheal disease in weanling rabbits than that resulting from either pathogen alone.

Hypervitaminosis A and reproductive disorders in rabbits.

Reproductive abnormalities in New Zealand White rabbits at a large commercial rabbitry were linked to an excess of dietary retinyl acetate. Fetal resorptions, abortions, and stillbirths were common in pregnant does. Examination of aborted and stillborn fetuses disclosed hydrocephalus, microencephaly, and cleft palate. Analysis of the commercially prepared feed disclosed a total vitamin A content of 102,278 IU/kg, of which 97,618 IU was retinyl acetate (recommended total vitamin A concentrations are 6,000 to 12,000 IU/kg). Levels of vitamin A in the plasma of does with reproductive disorders were 517 to 1,667 ng/ml (normal level is 300 ng/ml), and liver levels were 2,070 to 12,854 micrograms/g (normal range is 50 to 300 micrograms/g).

Pathogenicity of rotavirus in rabbits.

The role of rotavirus in diarrheal disease of rabbits was investigated, and a model for human rotavirus infection was established. Orogastric inoculation of 8- and 12-week-old New Zealand White rabbits with a rabbit strain of rotavirus (L:ALA:84) resulted in fecal shedding of virus for 6 to 8 days from 2 to 5 days after inoculation. Most rabbits exhibited diarrhea, coincident with the onset of viral shedding, which persisted for 2 to 4 days. Diarrhea was characterized by soft or fluid stools and fecal staining of the perineum. Inoculation of 3-week-old rabbits resulted in a briefer period of viral shedding and diarrhea of a milder nature. Histopathologic examination during the period of viral shedding revealed a mild, nonsuppurative enteritis. Inoculated rabbits exhibited antibodies in serum to rotavirus by enzyme-linked immunosorbent assay. Sham-inoculated or uninoculated rabbits maintained in the same cage or the same room with inoculated rabbits acquired rotavirus infection. The mild diarrheal disease which resulted with a rotavirus isolate from severe field cases suggests that cofactors were involved.

Pasteurella multocida and Bordetella bronchiseptica infections in rabbits.

The natural history of infection with Pasteurella multocida and Bordetella bronchiseptica in domestic rabbits was studied prospectively at a commercial rabbitry. At weaning, about 25% of rabbits had nasal infections with P. multocida and 75% had infections with B. bronchiseptica. Infection of weanling rabbits paralleled nasal infections of their dams. The proportion of rabbits with both infections increased with age. At 2 to 4 months old, about 50% of rabbits with P. multocida or P. multocida and B. bronchiseptica infections had upper respiratory disease (URD), whereas rabbits with B. bronchiseptica infection had no disease. In rabbits about 10 months old, 75% with P. multocida or P. multocida and B. bronchiseptica infections had URD, whereas virtually none with B. bronchiseptica infection had disease. Disease of the nares, paranasal sinuses, middle ears, and lungs was associated with P. multocida and not B. bronchiseptica infection. In adult rabbits with nasal P. multocida infection, with or without signs of URD, about 80% had concurrent infection of the paranasal sinuses and middle ears and 20% had infection of the bronchi and lungs. In rabbits without nasal P. multocida infection, 20 to 35% had P. multocida infection of the paranasal sinuses and middle ears. Weanling rabbits with and without P. multocida infection had similar immunoglobulin G (IgG) levels. In rabbits observed prospectively, the only antibody differences between those transiently and persistently infected with P. multocida were a diminished IgA response in nasal lavages and an earlier IgM response in sera of transiently infected rabbits. IgG levels increased with the duration of infection. There was no relationship between immunoglobulin levels and freedom from P. multocida infection.

Prevalence of coronavirus antibodies in rabbits.

Antibodies to coronavirus were detected by an indirect fluorescent antibody test in rabbit sera from six rabbitries. The prevalence ranged from 3 to 40% in different rabbitries and most seropositive rabbits were more than 4 months old. A rabbitry with high prevalence of antibodies and high incidence of diarrhea could serve as a source of virus and aid in studying the natural history of coronavirus infection in rabbits.

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.

Field trial of a live streptomycin dependent Pasteurella multocida serotype A:12 vaccine in rabbits.

A live, streptomycin dependent, Pasteurella multocida (SDPM) serotype A:12 vaccine was evaluated for preventing pasteurellosis in two commercial rabbitries. Rabbits were inoculated intranasally at 5 weeks old with either 0.25 ml of vaccine containing 10(8) colony forming units/ml or 0.25 ml of diluent (control). A proportion of rabbits received a second intranasal inoculation 1 month later. Partial protection against P. multocida infection was observed 1 and 2 months after inoculation in rabbits given only one dose of vaccine. The incidence of clinical signs of pasteurellosis was similar in vaccinated and nonvaccinated market-age rabbits inoculated 4 to 6 weeks previously. In does maintained in the breeding colony, P. multocida infection and upper respiratory disease occurred more frequently in vaccinated than nonvaccinated rabbits. Humoral antibody responses (IgA, IgM, IgG) followed longitudinally were similar in vaccinated and nonvaccinated does. Hence, the SDPM vaccine was not efficacious in controlling P. multocida infection at these two rabbitries.