Role of Pasteurella granulomatis and Dermatobia hominis in the etiology of lechiguana in cattle.

Attempts were made to reproduce bovine lechiguana, a disease associated with Dermatobia hominis and Pasteurella granulomatis infections. Suspensions of Pasteurella granulomatis were mixed with each of the following: saponin, oil adjuvant, ground Dermatobia hominis, or 5% mucin. Each preparation was inoculated into 6 cattle. Twelve more cattle, 6 of which received dexamethasone, were inoculated with bacterial suspension alone. Abscesses but no lechiguana was produced in all 36 cattle. After abscess regression, 12 cattle were reinoculated with a suspension of mouse-passed P. granulomatis. Only abscesses were produced. The intralymphatic inoculation of P. granulomatis in 6 cattle did not produce the disease. Eleven cattle infected naturally with D. hominis had lesions containing dead larvae. These lesions were inoculated with P. granulomatis. Nine cattle were experimentally infected with larvae of D. hominis that had been contaminated with the bacteria. No lechiguana lesions were produced in these 20 cattle. Six cattle with severe natural D. hominis infection were inoculated in the larval lesions with P. granulomatis. One developed lesions indistinguishable from those of natural lechiguana. The lesions regressed after treatment with chloramphenicol. D. hominis larvae and exudate from lesions caused by the fly were collected from 7 cattle on 3 farms and examined bacteriologically. P. granulomatis was isolated from the larvae and the exudate of a healthy calf from a farm where lechiguana had never been observed. These results suggest that P. granulomatis has a causal role in lechiguana, and that D. hominis may be a carrier of the bacterium. These observations suggest that lechiguana occurs when severe D. hominis lesions are infected with P. granulomatis. The apparent long incubation period, the negative results obtained in the other experiments, and also the infrequent occurrence of the natural disease suggest that lechiguana is a disease for which Koch's postulates are not easily fulfilled.

Toxin production by Pasteurella granulomatis.

Pasteurella granulomatis (Pg) is a recently identified bacterium associated with proliferative fibrogranulomatous panniculitis (also called "lechiguana") in Brazilian cattle. Recent attempts to experimentally reproduce this disease have only been partially successful. We hypothesized that Pg may produce hemolysin(s) and/or cytotoxin(s) which could contribute to its pathogenicity in susceptible cattle. The objective of this study was to determine the presence and degree of hemolytic and leukotoxic activity of selected isolates of Pg. Either ovine or bovine blood agar plates were streaked with 1 of 7 Pg isolates, incubated at 37 degrees C +/- 1 C for 48 hours, and examined for hemolysis. Two of seven isolates showed hemolytic activity on bovine plates, while all seven showed hemolytic activity on ovine plates. By use of the CAMP reaction, involving simultaneous intersecting cultures of Staphylococcus aureus and Pg, all seven Pg isolates showed enhanced (positive CAMP) hemolysis within 24 hours on bovine blood agar plates. Preliminary results using tetrazolium (MTT) dye reductions with bovine neutrophils showed leukotoxicity in 13 of 16 Pg cultures. Alamar blue tests indicate leukotoxic activity for all 7 Pg isolates. We conclude that some Pg isolates have variable hemolytic and/or leukotoxic properties and that this variability (presence and/or degree) of these 2 properties may affect the relative pathogenicity of Pg in susceptible cattle.

A streptomycin-dependent live pasteurella multocida type-3 vaccine for the prevention of fowl cholera in turkeys.

A live streptomycin-dependent Pasteurella multocida (Serotype 3) vaccine was found to protect turkeys orally and parenterally against homologous challenge.

Demonstration of bacteriocin activity in bovine and bison strains of Pasteurella multocida.

Of 33 strains of Pasteurella multocida examined, 14 showed bacteriocin activity and 17 were susceptible to bacteriocin. The activity was increased by about twofold if the cultures were induced with ultraviolet radiation; however, no increase in bacteriocin activity was observed if the potential producer strains were induced with mitomycin C. The bacteriocin activity of potential producer strains was increased if CaCl2 was incorporated in the medium. The patterns of bacteriocin susceptibility indicate that these substances may ultimately contribute to a typing scheme for the species. An extra-chromosomal genetic element was not detected when a potential producer strain was not detected when a potential producer strain was tested by the dye-buoyant density gradient method. This fact suggests that the genetic material responsible for bacteriocin activity in P multocida is located on the host chromosome proper.

Improved method for obtaining streptomycin-dependent mutants from Pasteurella multocida and Pasteurella haemolytica, using N-methyl-N'-nitro-N-nitrosoguanidine.

N-Methyl-N'-nitro-N-nitrosoguanidine was used in a modified and improved procedure to obtain streptomycin-dependent mutants of type A Pasteurella multocida and type 1 Pasteurella haemolytica. The relative virulence of these mutants was determined by mouse inoculation with and without streptomycin.

Live streptomycin-dependent Pasteurella multocida vaccine for the prevention of hemorrhagic septicemia.

A type B Pasteurella multocida was used for the development of a streptomycin-dependent (StrD) vaccine. Pasteurella multocida R-473, a hemorrhagic septicemia strain, was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine to increase the likelihood of encountering a StrD mutant and was plated on agar containing 400 microgram of streptomycin/ml. Replica plating was used to differentiate dependent from resistant colonies. Mice and rabbits were vaccinated with a StrD mutant and 21 days later, were challenge exposed, along with unvaccinated controls, with the wild type R-473. Protection of greater than 4 logs was shown for the vaccinated mice. All vaccinated rabbits were protected and all nonvaccinated controls succumbed to a challenge exposure of 500 or 1,000 LD50.

Hemoglobin enhancement of experimental infection of mice with Pasteurella haemolytica.

A 2% hemoglobin preparation injected into mice along with Pasteurella haemolytica culture was found to be as good an enhancer of virulence as a 7% swine gastric mucin preparation. The hemoglobin preparation was easier to prepare and was less toxic than mucin for mice when injected intraperitoneally.

Multiple drug resistance in Pasteurella multocida and Pasteurella haemolytica from cattle and swine.

The results of antimicrobial susceptibility testing on 262 strains of Pasteurella multocida and 141 strains of Pasteurella haemolytica isolated from cattle and swine from 1971 to 1974 were analyzed for patterns of resistance to streptomycin, penicillin, tetracycline, and chloramphenicol, using a modified Kirby-Bauer procedure. Resistance was recorded for 80.5% of the isolants of P multocida and 92.2% of those of P haemolytica. Resistance to streptomycin was most frequent, followed by resistance to penicillin and tetracycline. Most cultures of P multocida and P haemolytica were susceptible to chloramphenicol. There were 9 patterns of resistance with the aforementioned antibiotics. The combinations, streptomycin and penicillin and streptomycin and tetracycline, each accounted for approximately 10% of the resistance patterns of P multocida. Approximately half of the 14 isolants of P haemolytica were resistant to the combination of streptomycin, penicillin, and tetracycline. These observations underscore the need for antimicrobial susceptibility testing of clinical isolants of P multocida and P haemolytica.

Prevention of haemorrhagic septicaemia in buffaloes and cattle with a live vaccine.

Young cattle and buffaloes were vaccinated subcutaneously and intradermally with a live vaccine containing Pasteurella multocida serotype B:3,4. Twelve months after vaccination three of five young cattle in the subcutaneously vaccinated group and three of four in the intradermally vaccinated group were protected against serotype B:2 challenge. Eleven buffaloes vaccinated subcutaneously and two vaccinated intradermally survived the same challenge 13 months after vaccination.

Preliminary field trails with a streptomycin-dependent vaccine against haemorrhagic septicaemia.

In two trials, 68 cattle and buffalo calves, four to 10 months of age, were vaccinated with a vaccine containing a live streptomycin dependent mutant of Pasteurella multocida type B, isolated in Sri Lanka. Immunity was assessed using the passive protection test in mice with cattle and buffalo sera before and two or three weeks after vaccination. A single dose of vaccine conferred immunity in 66.6 to 83.3 per cent of cattle and 100 per cent of buffalo calves. A booster dose given three weeks later enhanced the immunity in cattle. There was no significant difference in the response, whether the vaccine was administered by the subcutaneous or intramuscular route. No adverse reactions were observed in any of the vaccinated animals.

Prevention of experimental haemorrhagic septicaemia with a live vaccine.

Pasteurella multocida serotype B:3,4 isolated from a fallow deer in England was used as a vaccine to prevent haemorrhagic septicaemia. The deer strain was less virulent for calves than typical serotype B:2 of haemorrhagic septicaemia strains. It elicited antibodies in cattle that protected mice against serotype B:2 infection. The live deer vaccine containing 2 X 10(7) viable organisms per dose was used to immunise calves. Six months after vaccination, five of six calves were protected against serotype B:2 challenge. Two calves challenged nine months after vaccination survived the same challenge. The live vaccine was more efficacious than an alum precipitated vaccine in protecting calves against B:2 challenge.

Identification of serotypes of Haemophilus pleuropneumoniae by counterimmunoelectrophoresis.

Counterimmunoelectrophoresis, direct immunofluorescence and immunodiffusion procedures were used to serotype 15 strains of Haemophilus pleuropneumoniae isolated from the respiratory tract of pigs in southern Brazil. Antigens were prepared by extracting cultures with a saline solution or by the phenol-water method. Antisera were prepared in rabbits against serotypes 1, 2, 3 and 5. Thirteen of the isolates were type 5 and two were type 3. No differences were observed between the results obtained in serotyping with counter immunoelectrophoresis and direct immunodiffusion, but both procedures were significantly better than immunodiffusion except with the saline extracted antigen. Counterimmunoelectrophoresis was quicker, more sensitive and more easily performed than the other techniques.

Lechiguana (focal proliferative fibrogranulomatous panniculitis) in cattle.

Lechiguana is a disease of cattle characterized by large, hard, subcutaneous swellings that grow rapidly and result in death after 3-11 months in untreated animals. Cattle treated with antibiotics recover. The disease has been reported from five states in south and southeastern Brazil. Histologically, the lesion consists of focal proliferation of fibrous tissue infiltrated by plasma cells, eosinophils, lymphocytes and sometimes neutrophils. The primary lesion is an eosinophilic lymphangitis, which results in eosinophilic abscesses, with occasional rosettes containing bacteria in their centres. Much experimental and epidemiological evidence, reviewed in this article, supports the suggestion that lechiguana is caused by an association of Pasteurella granulomatis (syn: Mannheimia granutomatis) and Dermatobia hominis.