Ultrastructural pathology of the upper respiratory tract of rabbits experimentally infected with Pasteurella multocida A:3.

Twenty-four 8 to 9 week-old Pasteurella multocida -free rabbits were divided into three equal groups, the first group was pretreated with hydrocortisone and inoculated intranasally with pasteurella multocida serotype A:3. The second group was inoculated intranasally with P. multocida without hydrocortisone treatment. The third group was inoculated with phosphate buffered saline only and used as a control group. Pasteurella multocida was isolated from the nasal cavity of all infected rabbits in group 1 and 2 and from the trachea of seven rabbits in group 1 and five rabbits in group 2. This study was conducted to observe the ultrastructural changes of the upper respiratory tract of hydrocortisone treated and non-treated rabbits infected with P. multocida serotype A:3. The ultrastructural changes detected in infected rabbits were ciliary destruction and deciliation of the ciliated epithelial cells, cellular swelling, goblet cell hyperplasia and endothelial cell damage. Pasteurella multocida was observed attached to the degenerated cilia, microvilli and mucus. Pasteurella multocida infection was associated with inflammatory responses, which may have caused tissue damage. It is possible that hydrocortisone modulates the severity of infection as an immune suppressor and an inhibitor of goblet cell secretion.     

Detection of antibodies against Pasteurella multocida using immunohistochemical staining in an outbreak of rabbit pasteurellosis

To detect serum antibody against Pasteurella multocida (P. multocida) in infected rabbits. a modified immunoperoxidase assay was applied. An outbreak of P. multocida infection in rabbits started from sudden death. The infected rabbits had severe fibrinous and purulent pneumonia with hemorrhage, and a large number of P. multocida (A:12) was isolated from the trachea and lungs of the animals. Antibodies of IgM and IgG to P. multocida were assessed by immunohistochemical staining using the sera of the animals as primary antibodies and applying them to formalin-fixed, paraffin-embedded sections of P. multocida attached to calf fibrin. IgM antibodies to P. multocida were first detected 7 days after the onset of the disease. IgG antibodies began to rise on the 7th or 14th day. These results suggested that the modified immunoperoxidase assay could detect antibodies against P. multocida.     

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 na?ve 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.     

Occurrence of Pasteurella multocida and related species in village free ranging chickens and their animal contacts in Tanzania

Investigation was done to determine the presence of Pasteurella multocida and related species in free ranging chickens and ducks, dogs, cats and pigs in three climatic zones (cool, warm and hot) of rural Morogoro, Tanzania. A total of 153 isolates of P. multocida ssp. multocida and related species were obtained by direct culture on blood agar, selective medium and mouse inoculation. P. multocida ssp. multocida was isolated from 0.7% of chickens and 7% of ducks. In dogs and cats, P. multocida ssp. multocida was isolated from 1 and 68%, respectively. One isolate of Pasteurella gallinarum was isolated from a duck. Other species obtained were; P. multocida ssp. septica, Pasteurella stomatis and taxon 16 from dogs and cats, while Pasteurella dagmatis and Pasteurella canis were found in dogs only. Prevalence of P. multocida ssp. multocida was significantly higher (P<0.01) in ducks of the warm zone (22%) than in ducks of other zones (0%). No significant difference was observed between the prevalence of P. multocida ssp. multocida in chickens of the warm zone (2%) and chickens of the cool and hot zones (0%). Extended phenotypic characterization revealed phenotypic similarities between two isolates from chickens and the duck strains. Mouse inoculation appeared to be more sensitive in detecting P. multocida ssp. multocida than blood agar and selective medium. Direct culture on blood agar recovered most of the isolates from dogs. This study has demonstrated for the first time the presence of P. multocida and related species in the village free ranging chickens, ducks, dogs and cats in Tanzania. Other non-classified Pasteurella spp. were also observed in the study, but further characterization is required before the final classification can be made. This paper reports for the first time the isolation of unclassified Pasteurella from dogs and cats in Africa. The results implies that fowl cholera might be occurring in free ranging poultry, and dogs and cats kept in contact might serve as sources of P. multocida to chickens and ducks. Subsequent applications of molecular techniques to analyse the epidemiological relatedness of clones isolated from different host species is indicated.     

Transmission of Pasteurella multocida on California turkey premises in 1988-89.

Restriction endonuclease analysis (REA) of whole-cell DNA was used to determine possible sources of Pasteurella multocida for each outbreak of fowl cholera occurring in turkey flocks in eight commercial poultry companies in California from October 1988 to September 1989. Over this period, 179 isolates of P. multocida were obtained from dead turkeys in 80 meat and breeder flocks on 43 premises. P. multocida was isolated from wildlife on five premises. Isolates were characterized by subspecies, serotype, presence of plasmid DNA, and REA type. In 52 (65%) flocks, all isolates of P. multocida had the same REA pattern as the M9 live vaccine strain following digestion of DNA with the restriction enzyme SmaI. Field strains of P. multocida were obtained from 27 (34%) flocks, and one flock (1%) yielded both M9 and a field strain of the organism. REA of field strains of P. multocida revealed 17 different SmaI REA types. Based on matching SmaI REA types, potential sources of P. multocida were identified for 15 of the 28 flocks infected with field strains of the organism, and transmission between turkey premises was a possibility in only seven flocks.     

A survey of potential virulence markers from avian strains of Pasteurella multocida

Twenty-four isolates of Pasteurella multocida from clinical cases of fowl cholera and the Clemson University vaccine strain were surveyed for the presence of potential virulence markers. Membrane proteins, enzymatic activity of the membrane proteins, and carbohydrate fermentation patterns were also determined to demonstrate phenotypic relationships within the groups. Few differences were found in these phenotypic characteristics among the isolates. Almost all the organisms produced siderophore and were hemolytic on turkey red blood cells. No extracellular enzyme or bacteriocin activity was detected and little antibiotic resistance was found. However, many organisms contained plasmids and demonstrated some degree of resistance to complement. Both characteristics were correlative markers in Pasteurella multocida isolated from birds with fowl cholera.     

10日龄肉鸭混合感染新型鸭肝炎病毒和多杀性巴氏杆菌的病原学研究

对1例疑似鸭肝炎病毒和多杀性巴氏杆菌混合感染的10日龄肉鸭采用常规的病毒、细菌鉴定方法和RT-PCR、PCR方法分别进行病毒、细菌的分离与鉴定。病毒鉴定为新型鸭肝炎病毒,细菌鉴定为荚膜血清A型多杀性巴氏杆菌多杀亚种。细菌对SPF鸡的毒力试验结果显示,分离的巴氏杆菌与强毒标准株C48-1毒力相近,为强毒株。细菌对10日龄肉鸭的致病性回归试验结果表明,一定数量的该株巴氏杆菌可导致10日龄雏鸭的感染死亡。结果表明,该批肉鸭为新型鸭肝炎病毒和A型多杀性巴氏杆菌混合感染。这是国内首例从感染鸭肝炎病毒10日龄雏鸭肝脏中分离到多杀性巴氏杆菌。     

Dermal necrosis caused by Pasteurella multocida infection in turkeys

Severe dermal necrosis caused by Pasteurella multocida Serotype 1 was diagnosed in three dressed turkey carcasses and two live turkeys from a commercial flock. The dressed carcasses were among several condemned at a processing plant. The isolate, P. multocida Serotype 1, produced progressive dermal necrosis when experimentally inoculated into injured skin of turkeys. The organism was reisolated from the dermal lesions. The turkey houses were found to be infested by mice; the skin injury and infection with P. multocida probably originated from mouse bites.     

Molecular epidemiology of Pasteurella multocida in turkeys.

Pasteurella multocida isolated from turkeys during an outbreak of fowl cholera was characterized by serotype and heterogeneity of genes encoding rRNA (ribotype) to investigate the epidemiology of the organism. Isolates were collected between October 1985 and July 1986. The M9 or Clemson University fowl cholera vaccine-like strain was detected in 17% of the flocks with fowl cholera. One particular strain, isolated only from breeder flocks, was recovered from 7 of the 10 breeder flocks examined in this study. Intracompany transmission appeared to be common, implying a failure in biosecurity. Circumstantial evidence indicated that in the field; the incubation period of P multocida in a turkey flock may be between 2 to 7 weeks. Wildlife did not appear to be an important reservoir of P multocida for turkeys during this study period. Ribotyping results tended to discount several of the possible interflock transmissions, as suggested by examination of serotyping results alone; however, serotyping in combination with ribotyping proved helpful in understanding the epidemiology of P multocida in turkeys.     

Virulence of avian capsular serogroup B Pasteurella multocida for turkey poults

Two strains of capsular serogroup B Pasteurella multocida isolated from avian hosts (swan and turkey) were evaluated for virulence based on lethality for turkey poults. Groups of poults were exposed intramuscularly to various concentrations of organisms of each strain. Both strains were virulent. The strain isolated from a turkey was highly virulent: all exposed poults died in less than 24 hours, including those exposed to only 79 organisms. This highly virulent strain was neither highly invasive nor highly infective: intrapharyngeal exposure with 7.9 x 10(6) organisms resulted in death of only one of five poults, and attempts to isolate the organism from pharyngeal mucosae and livers of surviving poults were unsuccessful. The high degree of virulence of a B capsular group strain isolated from a turkey indicates a disease-producing potential for members of this uncommon serogroup of P. multocida.     

Modulation of cross-protection factor(s) of avian Pasteurella multocida

Cross-protection factor(s) (CPF) of Pasteurella multocida were maintained in vitro through at least 9 serial passages. Different growth media and temperatures enhanced or repressed the ability of P. multocida to produce CPF. Certain amino acids were innoculous to expression of CPF. B-vitamins enhanced CPF, whereas certain inorganic salts repressed CPF. The plasma of normal tuekeys contained a compound or compounds that were responsible for expression and maintenance of CPF.     

Pasteurella multocida-associated sinusitis in khaki Campbell ducks (Anas platyrhynchos)

Pasteurella multocida group B, serotype 3, was isolated from sinusitis-affected khaki Campbell ducks. To study the role of P. multocida in sinusitis, commercial khaki Campbell ducks were experimentally infected with P. multocida alone or combined with Escherichia coli. In Expt. 1, experimental ducks were infected with P. multocida intranasally or ocularly. A comparison was done by intranasal inoculation with pooled nasal discharge from the affected ducks or phosphate-buffered saline. The ducks intranasally inoculated with the nasal discharge or P. multocida showed sinusitis. In Expt. 2, E. coli alone or a combination of P. multocida and E. coli was intranasally inoculated into experimental ducks. The ducks intranasally inoculated with the combination of P. multocida and E. coli had sinusitis, the same as found in the field but less severe than that of the field cases. Pasteurella multocida was already present in litter/floor of duck farms. We concluded that P. multocida played a role in induction of sinusitis. However, the sinusitis in ducks may be initiated by poor management, especially in the brooding period of ducks.     

Septicemia in vaccinated and nonvaccinated turkeys inoculated with Pasteurella multocida serotype A:3,4

Sixty-four, 10-week-old turkeys were inoculated with a highly virulent field isolate (86-1913) of Pasteurella multocida serotype A:3,4 by an oculo-nasal-oral route. Inoculated turkeys were examined at 4, 8, 16, 20, and 24 hours post-inoculation for bacteremia and histologic lesions. Bacteremia was detected in one of six turkeys 8 hours after inoculation and in four of six turkey poults at 16 hours post-inoculation. Pasteurella multocida was isolated from the spleens of two turkeys at 8 hours and from the spleens of all six poults 16 hours after inoculation. Peak concentrations of P. multocida reached 10(9) colony forming units per ml of blood. At 4 to 8 hours post-inoculation, isolate 86-1913 produced a fibrinopurulent bronchopneumonia followed by severe pulmonary necrosis, pleuritis, vasculitis; and, at 16 to 24 hours post-inoculation numerous extracellular bacteria were observed. Hepatic lesions included focal heterophil aggregates 8 hours after inoculation; these progressed to hepatic necrosis. Numerous extracellular bacteria within sinusoids were present 16 to 24 hours after inoculation. At 16 to 24 hours post-inoculation, there was degeneration of periarteriolar reticular cells in the spleen; these cells progressed to coalescing coagulative splenic necrosis with extracellular bacterial colonies. A second group of 41, 10-week-old turkeys, previously vaccinated with the Clemson University strain of P. multocida serotype A:3,4, were challenged with isolate 86-1913.(ABSTRACT TRUNCATED AT 250 WORDS)     

Colonization of the pharyngeal tonsil and respiratory tract of the gnotobiotic pig by a toxigenic strain of Pasteurella multocida type D

Seven-day-old gnotobiotic pigs were inoculated intranasally with Pasteurella multocida and euthanatized 2, 5, 9, and 14 days after inoculation. Tissues from the oropharynx and respiratory tract of pigs were cultured quantitatively and analyzed microscopically. Pigs remained afebrile and alert, except one that died of acute fibrinopurulent pneumonia. Pasteurella multocida was isolated in greatest numbers from the pharyngeal tonsils, but only in low numbers from turbinate, trachea, lung, spleen, and liver. Significant histologic changes were limited to the tonsil. Infected pigs developed mild tonsillitis with lymphocytic hyperplasia, and accumulation of cell debris and bacteria in crypts. Capsular antigens of P. multocida, identified on tissue sections with rabbit anti-capsular polysaccharide antibody and immunocytochemical reagents, were confined to the crypt lumen. Ultrastructurally, bacteria were free within crypt material or within phagosomes of macrophages or neutrophils. In a second experiment, 5-day-old pigs were infected with Streptococcus suis type 2, followed by toxigenic Pasteurella multocida at 7 days of age; one pig died of streptococcal septicemia. Pigs developed a mild tonsillitis, and both bacteria were cultured from the tonsillar crypts for up to 14 days after infection. These studies show that a toxigenic strain of Pasteurella multocida, which is a causative agent of atrophic rhinitis, can colonize the tonsil and respiratory tract of gnotobiotic pigs for up to 14 days. In addition, colonization can occur concurrently with Streptococcus suis type 2.     

Characterization of toxin from different strains of Pasteurella multocida serotype A and D

Chromosomal DNA from 13 different selected Pasteurella multocida spp. multocida strains of serotypes A and D were isolated and compared. All 10 toxigenic strains were recognized by a DNA probe which included the toxA gene coding for the Pasteurella multocida toxin (PMT). None of the three nontoxigenic strains reacted with the DNA probe. Toxin from the 10 toxigenic strains were isolated and compared. All were found to possess the biological characteristics previously described for the PMT isolated from P. multocida ssp. multocida NCTC 12178, including molecular mass of approx. 143 kDa and reactivity with a series of monoclonal antibodies. Toxin prepared from different toxigenic strains could not be differentiated immunologically by tandem crossed immunoelectrophoresis, Toxin, which was affinity purified from four of the strains and subsequently inactivated by formaldehyde, was cross-protective when used for vaccination of mice before challenge with PMT. It is concluded that the toxin from toxigenic strains of P. multocida ssp. multocida must be very similar, if not identical.     

Restriction endonuclease analysis of Pasteurella multocida isolates from three California turkey premises.

Three California turkey premises that had repeated outbreaks of fowl cholera were studied for periods of 2 to 4 years. Using biochemical, serologic, plasmid DNA, and restriction endonuclease analyses of isolates of Pasteurella multocida from turkeys and wildlife on the premises, strains of the organism were found to be enzootic on two of the premises. On the third, a variety of strains of P. multocida were isolated from fowl cholera outbreak flocks.     

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.     

Comparison of enzyme-linked immunosorbent assay and indirect hemagglutination test for quantitating antibody responses in chickens against Pasteurella multocida

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to measure humoral antibody responses of chickens against Pasteurella multocida. A standard indirect hemagglutination (IHA) test was used to compare serologic results with those of ELISA. The ELISA was also used following challenge with P. multocida to compare the efficacy of three commercial fowl cholera vaccination regimens. Although antibody titers measured by ELISA and IHA were highly correlated, ELISA was at least twice as sensitive as IHA. Antibody measured by ELISA and IHA also correlated significantly with protection against P. multocida challenge. No mortality occurred in any of the three vaccinated challenged groups. However, control unvaccinated chickens experimentally infected with P. multocida developed signs of acute pasteurellosis and died by the 10th day post-challenge. Impression smears made of hepatic tissue from all chickens were stained (Wright's stain), and typical bipolar rods characteristic of Pasteurella were identified in smears from unvaccinated challenged controls only.     

Pharmacokinetics of penicillin G in the turkey

Parmacokinetics of penicillin G was determined for the turkey. The study was prompted by the isolation of a sulfonamide-resistant strain of Pasteurella multocida from tissues of turkeys involved in an outbreak of fowl cholera and the subsequent discovery that little pharmacologic information was available concerning other antimicrobial agents in that species. Penicillin G was chosen for study because P multocida is susceptible to this antibiotic. The elimination of the antibiotic followed first-order kinetics, and the half-life was found to be 0.5 hours. Parenteral administration of benzathine-procaine penicillin G resulted in higher concentrations, which persisted for longer periods than did procaine or potassium salts of the antibiotic.     

Comparison of methods for the sampling and isolation of toxigenic Pasteurella multocida from the nasal cavity of pigs

The efficacy of detecting toxigenic Pasteurella multocida from nasal swabs of slaughtered and live pigs was assessed. The isolation of toxigenic P multocida from nasal cavities of slaughtered bacon pigs from two herds with atrophic rhinitis was reduced by immersion in the hot water tank by 25 per cent and 75 per cent. Individual sows from one of the infected herds were repeatedly swabbed to find the best method of isolating toxigenic P multocida. Toxigenic P multocida were isolated from 50 per cent of cotton swabs inoculated on to selective medium the same day. After 24 hours in the post, 45 per cent of cotton swabs placed in transport medium, 38 per cent of alginate swabs dissolved in transport medium and inoculated into mice, and 36 per cent of the dissolved swabs inoculated directly on to selective medium yielded toxigenic P multocida. These bacteria were isolated from only 25 per cent of cotton swabs held in transport medium at 10 degrees C for 48 hours to simulate prolonged postage times; from slaughtered pigs a similar reduction in isolation was seen with swabs kept for 24 or 48 hours. The reduced isolation caused by a delay before culture was associated with an overgrowth of other flora. The development of this flora was prevented by storage of swabs at 4 degrees C in the laboratory or by the use of cool boxes for postage.