Epidemiologic investigation of Riemerella anatipestifer in a commercial duck company by serotyping and DNA fingerprinting.

A commercial duck company that raises approximately two million Pekin ducks per year experienced an outbreak of Riemerella anatipestifer (RA) on nine farms over a 1-yr period. Owing to concerns that the bacteria was being spread from farm to farm, an investigation using serotyping and DNA fingerprinting was performed. The results revealed that there were three different strains of RA involved in the outbreak. One strain was spread from one farm to six other farms, while another strain from the same farm was spread to two other farms. These findings add additional proof of the value of DNA fingerprinting in disease outbreak investigations and further support the importance of implementing biosecurity protocols to stop the spread of disease-causing organisms.

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 naïve 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.

Identification of new major histocompatibility complex class II restriction fragment length polymorphisms in a closed experimental line of Beltsville Small White turkeys.

Beltsville Small White (BSW) turkeys have been utilized as an experimental model in the study of bacterial, parasitic, and fungal diseases. Given the critical role of MHC antigens in the initial steps of the immune response to specific pathogens, the MHC Class II of BSW turkeys was characterized. Southern blot analysis of PvuII-digested turkey DNA that was hybridized with a chicken Class II beta gene genomic clone revealed two restriction fragment length polymorphism profiles not previously identified in experimental or commercial breeder lines of turkeys. These fingerprint profiles differed in a single 6.0-kb band that was present in approximately 38% of the birds examined. The DNA fragments of 5.0, 4.1, 3.3, and 3.1 were present in both profiles. Furthermore, no mixed lymphocyte reaction was observed between individuals within the BSW turkey line. The present results indicate that BSW turkeys represent a unique source of genetic diversity for MHC Class II haplotypes.

Effects of intranasal inoculation with Bordetella bronchiseptica, porcine reproductive and respiratory syndrome virus, or a combination of both organisms on subsequent infection with Pasteurella multocida in pigs.

OBJECTIVE: To determine effects of intranasal inoculation with porcine reproductive and respiratory syndrome virus (PRRSV) or Bordetella bronchiseptica on challenge with nontoxigenic Pasteurella multocida in pigs. ANIMALS: Seventy 3-week-old pigs. PROCEDURE: In experiment 1, pigs were not inoculated (n= 10) or were inoculated with PRRSV (10), P. multocida (10), or PRRSV followed by challenge with P. multocida (10). In experiment 2, pigs were not inoculated (n = 10) or were inoculated with B. bronchiseptica (10) or PRRSV and B. bronchiseptica (10); all pigs were challenged with P. multocida. Five pigs from each group were necropsied 14 and 21 days after initial inoculations. RESULTS: Pasteurella multocida was not isolated from tissue specimens of pigs challenged with P. multocida alone or after inoculation with PRRSV. However, in pigs challenged after inoculation with B. bronchiseptica, P. multocida was isolated from specimens of the nasal cavity and tonsil of the soft palate. Number of bacteria isolated increased in pigs challenged after coinoculation with PRRSV and B. bronchiseptica, and all 3 agents were isolated from pneumonic lesions in these pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Infection of pigs with B. bronchiseptica but not PRRSV prior to challenge with P. multocida resulted in colonization of the upper respiratory tract and tonsil of the soft palate with P. multocida. Coinfection with PRRSV and B. bronchiseptica predisposed pigs to infection of the upper respiratory tract and lung with P. multocida. Porcine reproductive and respiratory syndrome virus and B. bronchiseptica may interact to adversely affect respiratory tract defense mechanisms, leaving pigs especially vulnerable to infection with secondary agents such as P. multocida.

Ultrastructural location of the cross-protection factor on in vivo and in vitro grown Pasteurella multocida.

Pasteurella multocida from infected turkey tissues expresses a unique immunogen called cross-protection factor (CPF) that induces immunity to challenge by both homologous and heterologous serotypes. In this study, we used a monoclonal antibody (AMP MAb) to CPF and protein A-colloidal gold (PACG) to locate CPF on P. multocida. After incubation with AMP MAb and PACG, CPF was detected at the bacterial surface and cell periphery of P. multocida in infected turkey liver and P. multocida isolated from infected turkey blood. CPF was not detected on P. multocida incubated with control monoclonal antibody. Pasteurella multocida isolated from infected turkey blood and cultivated in the peptone-based medium did not express CPF consistently, and some cells contained more CPF than others. The location of CPF also varied, and CPF was detected both intracellular and extracellular on the cell surface. In the latter cells, CPF was heavily concentrated to a specific lateral site or detected sloughing from the cell surface. These results correlate with laboratory observations that CPF detected on P. multocida from infected turkey tissues, P. multocida isolated from infected turkey blood, and P. multocida cultivated in peptone-based medium is associated with outer membrane fractions.

Septicemic pasteurellosis in free-ranging neonatal pronghorn in Oregon.

As part of a study to determine the cause(s) of population decline and low survival of pronghorn (Antilocapra americana) neonates on Hart Mountain National Antelope Refuge (HMNAR), Oregon (USA), 55 of 104 neonates captured during May 1996 and 1997 were necropsied (n = 28, 1996; n = 27, 1997) to determine cause of death. Necropsies were conducted on fawns that died during May, June, or July of each year. The objectives of this study were to report the occurrence and pathology of pasteurellosis in neonates and determine if the isolated strain of Pasteurella multocida was unique. Septicemic pasteurellosis, caused by P. multocida, was diagnosed as the cause of death for two neonates in May and June 1997. Necropsy findings included widely scattered petechial and ecchymotic hemorrhages found over a large portion of the subcutaneous tissue, meninges of the brain, epicardium, skeletal muscle, and serosal surface of the thorasic and abdominal cavities. Histological examination of lung tissues revealed diffuse congestion and edema and moderate to marked multifocal infiltrate of macrophages, neutrophils, and numerous bacteria within many terminal bronchioles and alveoli. Pasteurella multocida serotypes A:3,4, and B:1 were isolated from several tissues including lung, intestinal, thorasic fluid, and heart blood. Each B:1 isolate had DNA restriction endonuclease fingerprint profiles distinct from isolates previously characterized from domestic cattle, swan (Olor spp.), moose (Alces alces), and pronghorn from Montana (USA). This is the first report of pasteurellosis in pronghorn from Oregon and the B:1 isolates appear to be unique in comparison to DNA fingerprint profiles from selected domestic and wild species.

Absence of protection against challenge with aspergillus fumigatus by adoptive transfer of splenocytes from convalescent turkeys.

Only limited protective immunity against aspergillosis after experimental immunization of turkeys has been previously demonstrated. No studies evaluating the efficacy of transfer of immunity in preventing aspergillosis in birds have been reported. This study consisted of two trials assessing the level of protection against Aspergillus fumigatus challenge afforded by transfer of splenocytes from convalescent turkeys. Three treatment groups of 12-to-14-wk-old Beltsville small white (BSW) turkeys comprising the splenocyte donors were prepared by one of the following: 1) intra-air sac (IA) challenge with A. fumigatus conidia 5 wk prior to transfer; 2) IA challenge and then intravenous (i.v.) injection of killed conidia 1 wk prior to transfer; or 3) sham inoculations. Splenocytes from each group were pooled, enriched for mononuclear leukocytes by density gradient centrifugation, and diluted in cell culture medium (CM). Cell viability was assessed by dye exclusion. Each splenocyte preparation was administered intravenously to one of three recipient groups consisting of 10 BSW turkeys each. A control group (n = 10) was given cell-free CM. Recipients were challenged with viable A. fumigatus conidia 16 hr after splenocyte transfer by unilateral IA (trial 1) or i.v. (trial 2) inoculation. Lesion scores postchallenge revealed no differences between turkeys given splenocytes from convalescent vs. naive (control) turkeys. IA exposure produced ipsilateral lesions in air sacs and lung, whereas i.v. exposure produced severe miliary hepatitis. Donor cell function was confirmed by mitogen blastogenesis; however, cells were nonresponsive to A. fumigatus antigens, regardless of previous exposure status.

Typing of Pasteurella multocida from haemorrhagic septicaemia in Danish fallow deer (Dama dama).

Isolates of Pasteurella multocida ssp. multocida (n = 31) from a Danish population of fallow deer which succumbed to haemorrhagic septicaemia during 1992 1993 and isolates from the palatine tonsils of apparently healthy fallow deer from the same area (n=6) were typed and compared with P. multocida from other sources. Plasmids were net observed in the fallow deer strains and one unique pattern was observed by ribotyping using HindIII and by pulsed-field gel electrophoresis using SanlI as restriction endonuclease. All Danish fallow deer isolates belonged to serotype B:3,4. On restriction endonuclease analysis using HhaI as restriction endonuclease, all had a profile identical to that of a fallow deer isolate from the United Kingdom: profile 0033 of Wilson et al. On restriction endonuclease analysis using HpaII as restriction endonuclease, the Danish fallow deer isolates had a unique profile, designated 0062, which differed slightly from that of a fallow deer isolate from the United Kingdom. P. multocida from other animal species were genotypically different from the fallow deer isolates. It is concluded that a specific clone of P. multocida was responsible for the outbreak of haemorrhagic septicaemia among Danish fallow deer. A carrier rate of 27% was demonstrated among apparently normal animals from the same population.

Hemorrhagic septicemia in fallow deer (Dama dama) caused by Pasteurella multocida multocida.

Four outbreaks of hemorrhagic septicemia caused by Pasteurella multocida multocida occurred in a population of 1,800 fallow deer (Dama dama) during 1992-1996. A total of 340 fallow deer were submitted for postmortem examination. Pasteurellosis was diagnosed in 273 of 312 deer suspected of having septicemia. Pasteurella multocida was isolated from 257 animals, and the diagnosis was based on typical pathologic changes alone in the other 16 animals. Pasteurella multocida was isolated in pure culture from 219 of 248 samples of cerebrospinal fluid. Eighteen animals were observed moribund with severe depression, foamy nasal discharge, and respiratory distress, and 257 were found dead. Major clinical signs and pathologic changes included extensive swelling of the head and the neck and peracute or acute septic pneumonia, petechial and ecchymotic hemorrhages on serous membranes, and severely hemorrhagic adrenal glands and abomasum. Rhinitis and necrotic pharyngeal mucosae were common. Histologically, the most advanced lesions were in the nasal mucosa and pharynx. The swelling of the head and the neck arose from a diffuse cellulitis in the subcutaneous and intermuscular tissues. The earliest lesions in the lungs included large numbers of bacteria in the pulmonary capillaries, but various degrees of fibrinous exudation to the alveoli and infiltration with heterophils usually were observed.

Enhanced adhesion of Pasteurella multocida to cultured turkey peripheral blood monocytes.

Capsular hyaluronic acid (HA) mediates adhesion of serogroup A strains of Pasteurella multocida to elicited turkey air sac macrophages (TASM). In contrast, freshly isolated turkey peripheral blood monocytes (TPBM) do not bind serogroup A strains. Following culture of TPBM for 6 days in chamber slides, adhesion of the bacteria to TPBM increased gradually. Incubation in chamber slides coated with entactin-collagen IV-laminin (ECL) attachment matrix or exposure to phorbol myristate acetate (PMA) further enhanced the adhesion of P. multocida to TPBM. Addition of HA, but not Arg-Gly-Asp peptide, to TPBM culture inhibited bacterial adherence similarly to the inhibition previously reported for TASM. Exposure of TPBM to monoclonal antibody directed against HA-binding cell surface proteoglycan (CD44) decreased binding of P. multocida. Collectively, these findings indicate that P. multocida adhesion to TPBM is mediated by capsular HA and can be increased by culture on ECL attachment matrix or PMA exposure. Additionally, the findings suggest that the capsular mucopolysaccharide of serogroup A strains of P. multocida recognizes an isoform of CD44 expressed on cultured TPBM.

Morphological effects of Pasteurella multocida type-D dermonecrotoxin on rat osteosarcoma cells in a nude mouse model.

Of 15 athymic nude mice that received subcutaneous implants of a rat osteosarcoma cell line, two groups of four subsequently received either a short (group 1) or a more prolonged (group 2) course of subcutaneous injections of the dermonecrotic toxin (DNT) of Pasteurella multocida type D. The remaining seven mice (controls) received no DNT. Both groups of DNT-treated mice lost body weight as compared with controls. Tumour weight, expressed as a percentage of body weight, increased in the four group 1 mice. Tumours in this group 1 were consistently larger than those in appropriate controls, indicating that this percentage was not simply a function of decreased body weight. The immunohistochemical labelling of proliferating cell nuclear antigen (PCNA) and morphometric analysis of intratumoral necrosis suggested that the DNT had a mitogenic effect and contributed to the neoplastic growth. The presence of foci of neoplastic osteoblasts in the lungs of some DNT-treated mice suggested that the enhanced tumour growth led to an increased incidence of metastasis.

DNA fingerprinting of Riemerella anatipestifer.

Seventeen restriction endonucleases were evaluated for use in DNA fingerprinting of Riemerella anatipestifer. Digestion of chromosomal DNA with either HinfI or DdeI restriction endonuclease, followed by submarine electrophoresis in agarose and staining with ethidium bromide, resulted in DNA fingerprint profiles that could be easily resolved. HinfI produced readable fingerprint patterns in the 2.7-20-kb range and was used to distinguish DNA fingerprint profiles among 89 strains of R. anatipestifer representing isolates from various avian species in the United States, the United Kingdom, Australia, Canada, Germany, and Israel. A total of 52 distinct DNA fingerprint profiles were found.

Bacterin-induced protection of turkeys against fowl cholera following infection with Bordetella avium.

Groups of Beltsville small white turkeys, passively immunized and not passively immunized against Bordetella avium, were challenged with live B. avium at 2 days of age. Birds not passively immunized developed severe bordetellosis with early onset, whereas passively immunized birds developed mild bordetellosis with late onset. Following convalescence, birds with and without exposure to B. avium were vaccinated against fowl cholera with a water-in-oil bacterin. The birds were given a homologous challenge with serotype A: 3 Pasteurella multocida. Although no difference in protection against fowl cholera was seen between vaccinated birds that were previously infected with B. avium and those that were not, survivability was better in birds given two doses rather than 1 dose of bacterin.

Early pulmonary lesions in turkeys produced by nonviable Aspergillus fumigatus and/or Pasteurella multocida lipopolysaccharide.

This study assessed the potential of lipopolysaccharide (LPS) purified from Pasteurella multocida to cause pulmonary pathology or exacerbate lesions produced by gamma-irradiated nonviable Aspergillus fumigatus conidia when administered via the intra-air sac route in turkeys. LPS provoked suppurative airsacculitis, pleuritis, and pneumonia. Nonviable conidia produced airsacculitis and transient pneumonitis but did not elicit multinucleate giant cells, which are a feature of the inflammatory process in A. fumigatus infection. LPS in combination with A. fumigatus conidia resulted in accelerated pulmonary inflammation and apparently delayed clearance of conidia from pulmonary tissues. This study presents a model of aseptic airsacculitis and pneumonia with clinical relevance.

Effects of Pasteurella multocida toxin on porcine bone marrow cell differentiation into osteoclasts and osteoblasts.

The effect of Pasteurella multocida toxin (PMT) on porcine osteoclast and osteoblast differentiation was studied using in vitro cell culture systems. When grown in the presence of Vitamin D3, isolated porcine bone marrow cells formed multinucleated cells with features characteristic of osteoclasts. Exposure of bone marrow cells to Vitamin D3 and PMT during growth resulted in formation of increased numbers and earlier appearance of osteoclasts compared to controls. Ultrafiltered medium form PMT-treated cells likewise increased osteoclast numbers, suggesting that a soluble mediator may be involved in the action of PMT. When cell cultures were treated with fluorescein-labeled PMT, fluorescence was found within the cytoplasm of small, round cells that did not resemble either osteoclasts or osteoclastic precursor cells. Cultures of porcine bone marrow cells exposed to dexamethasone, ascorbic acid, and beta-glycerophosphate developed into osteoblastic cells that formed multilayered, mineralized nodules. Exposure of osteoblastic cultures to low concentration of PMT resulted in retarded cell growth, formation of decreased numbers of nodules and minimal to no mineralization in the nodules; higher concentration of PMT resulted in increased cellular debris and poor growth of cells, with no nodule formation. These findings suggest that PMT may induce turbinate atrophy in pigs by increasing osteoclast numbers and inhibiting osteoblastic bone formation. The effect of PMT on osteoclastic differentiation and growth may not be due to a direct effect on preosteoclastic cells, but rather due to alterations in the soluble mediator secretion by marrow stromal cells.

Passive immune protection of turkeys against Coryza.

Antisera made against whole cells of Bordetella avium protected turkeys against disease signs of turkey coryza, but antiserum against the dermonecrotic heat-labile toxin (DHLT) did not. Neither antiserum against whole cells nor antiserum against DHLT protected turkeys against colonization of the trachea by B. avium. At least 25 bands in whole cell lysate of B. avium separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) reacted in immunoblots with protective antiserum. These bands occurred at < 40 kDa and > 105.3 kDa. DHLT had an isoelectric point (pI) in the range pH 6.3-6.7. Following SDS-PAGE of isoelectric-focused fractions, two bands were recognized by anti-DHLT with immunoblots of pI 6.3, pI 6.5, and pI 6.7 fractions separated by SDS-PAGE.

Pathology of acute aspergillosis in turkeys.

Pathologic changes were characterized by gross examination and light microscopy after intra-air sac inoculation of 9- and 19-wk-old turkeys with Aspergillus fumigatus conidia. Turkeys were euthanatized and examined at 24, 48, 72, and 96 hr postinoculation (PI). Lesions were largely confined to air sac membrane and lung tissues and were similar between the two age groups. There was progressive severity of gross lesions in both organs and of microscopic lesions in lung tissue. The character and severity of histologic lesions in air sac membranes were roughly similar at 24 through 96 hr PI, but there was an increasing amount and consolidation of exudate adherent to the epithelial surface. Lesions in air sac membranes included edema, heterophil and macrophage infiltrates, granulomas, lymphohistiocytic perivasculitis, necrosis, epithelial loss, and surface exudate. Discreet granulomas containing multinucleate giant cells were present at 24 hr PI and thereafter. Lung lesions progressed from mild interstitial pneumonia to extensive effacement by multifocal coalescing granulomas, necrosis, and hemorrhage. Severe pleuritis with local extension into lung parenchyma was seen at all time points. Numbers of intralesional fungal elements seen histologically were similar between age groups and appeared to decrease in air sac membranes and increase in lung tissues from 24 to 96 hr PI. Lung tissue of the 19-wk-old turkeys contained fewer colony-forming units per gram at time points after 24 hr PI.

Capsular hyaluronic acid-mediated adhesion of Pasteurella multocida to turkey air sac macrophages.

Serogroup A strains of Pasteurella multocida, the major cause of fowl cholera, are resistant to phagocytosis in nonimmunized birds. Adherence studies with a capsulated strain of P. multocida (serotype A:3) and turkey air sac macrophages in culture showed that the bacteria were capable of adhering in large numbers to the macrophages but were not internalized. A noncapsulated variant of the bacteria (serotype -:3) showed little or no adherence and was not internalized. These data indicated that the adhesive properties were caused by the presence of a capsule on the bacteria. The role of capsular hyaluronic acid in adherence to macrophages was investigated. Depolymerization of the bacterial capsule with hyaluronidase increased phagocytosis by macrophage cultures, and addition of hyaluronic acid to the macrophages inhibited bacterial adherence. Additionally, exposure of macrophages to chondroitin sulfate B, an anionic polysaccharide similar to hyaluronic acid, did not affect the adhesive properties and resistance to phagocytosis of capsulated organisms. Treatment of macrophages with sodium metaperiodate or trypsin suppressed bacterial binding. Collectively, these data indicate that P. multocida adhesion to air sac macrophages, but not internalization, is mediated by capsular hyaluronic acid and suggest that recognition of this bacterial polysaccharide is a result of a specific glycoprotein receptor.

Effect of Pasteurella multocida toxin on physeal growth in young pigs.

OBJECTIVE: To determine whether Pasteurella multocida toxin (PMT) affects growth of the proximal portion of the humerus of young pigs. ANIMALS: 20 colostrum-deprived, cesarean-derived pigs. DESIGN AND PROCEDURE: 5 groups (n = 4/group) of pigs were formed. Group-1 pigs received 0.1 ml of phosphate-buffered saline solution for 4 weeks; group-2 pigs received 0.05 microgram of PMT/kg of body weight at 14 and 21 days; group-3 pigs received 0.05 microgram of PMT/kg at 28 and 35 days; group-4 pigs received 0.1 microgram of PMT/kg at 14 and 21 days; and group-5 pigs received hyperimmune serum (from a sow given purified toxin) on days 13, 20, 27, and 34, and 0.1 microgram of PMT/kg on days 14, 21, 28, and 35. RESULTS: All pigs given 0.1 microgram of PMT/kg without serum died or were euthanatized, as were 4 pigs given 0.05 microgram of PMT/kg. These pigs had increased serum interleukin 1 and 6 bioactivities. Pigs surviving 0.05 microgram of PMT had decreased weight gain, rough coat, marked atrophy of the ventral concha (as determined by turbinate perimeter ratios), and small stature. The surviving pigs also had reduced area and decreased proliferation indices in physeal chondrocytes on the basis of bromodeoxyuridine immunoreactivity. Control and serum-treated pigs gained weight, had no clinical effects, had similar physeal areas, and had higher cell proliferation indices. CONCLUSIONS: PMT inhibits endochondral bone formation by reducing physeal area and chondrocyte proliferation in vivo. Hyperimmune serum neutralizes the effects of toxin on weight gain, clinical appearance, physeal area, and chondrocyte proliferation. CLINICAL RELEVANCE: PMT may affect growth of the skeletal system. Antiserum to PMT is protective.

Passive immune cross-protection in mice produced by rabbit antisera against different serotypes of Pasteurella multocida.

Haemorrhagic septicaemia (HS) and fowl cholera (FC) are specific diseases caused by certain serotypes of Pasteurella multocida. Strains that usually cause HS in cattle and water buffalo do not produce FC in avian species, and strains that cause FC do not produce HS in cattle and water buffalo. A variety of P. multocida serotypes, including unusual strains which can cause HS in wild ruminants, were evaluated in passive immune protection studies to determine the immunological relationship between strains associated with HS and FC. Various degrees of cross-protection were seen among the strains. Antiserum against a serotype B:3,4 strain protected against strains capable of causing HS (serotypes B:1, B:2, B:3,4, B:4 and E:2) and FC (serotypes A:1, A:3 and A:5). Antiserum against an FC strain (serotype A:5) similarly protected against strains capable of causing HS and FC. Antigenic analyses indicated that cross-protection was not necessarily induced by serotype-specific capsular (beta) or somatic (gamma) antigens or major porin protein. SDS-PAGE and immunoblots of whole cell lysates of the different HS and FC strains showed many protein-staining bands with similar mobilities and antigenic activity. These cross-reactive antigenic bands occurred in the 20- to 120-kDa range. Adsorption of antiserum with a heterologous serotype removed its reactivity with most of these bands, as well as its ability to cross-protect.