Interaction of Pasteurella multocida with free-living amoebae.

Pasteurella multocida is a highly infectious, facultative intracellular bacterium which causes fowl cholera in birds. This study reports, for the first time, the observed interaction between P. multocida and free-living amoebae. Amoebal trophozoites were coinfected with fowl-cholera-causing P. multocida strain X-73 that expressed the green fluorescent protein (GFP). Using confocal fluorescence microscopy, GFP expressing X-73 was located within the trophozoite. Transmission electron microscopy of coinfection preparations revealed clusters of intact X-73 cells in membrane-bound vacuoles within the trophozoite cytoplasm. A coinfection assay employing gentamicin to kill extracellular bacteria was used to assess the survival and replication of P. multocida within amoebae. In the presence of amoebae, the number of recoverable intracellular X-73 cells increased over a 24-h period; in contrast, X-73 cultured alone in assay medium showed a consistent decline in growth. Cytotoxicity assays and microscopy showed that X-73 was able to lyse and exit the amoebal cells approximately 18 h after coinfection. The observed interaction between P. multocida and amoebae can be considered as an infective process as the bacterium was able to invade, survive, replicate, and lyse the amoebal host. This raises the possibility that similar interactions occur in vivo between P. multocida and host cells. Free-living amoebae are ubiquitous within water and soil environments, and P. multocida has been observed to survive within these same ecosystems. Thus, our findings suggest that the interaction between P. multocida and amoebae may occur within the natural environment.

The pnhA gene of Pasteurella multocida encodes a dinucleoside oligophosphate pyrophosphatase member of the Nudix hydrolase superfamily.

The pnhA gene of Pasteurella multocida encodes PnhA, which is a member of the Nudix hydrolase subfamily of dinucleoside oligophosphate pyrophosphatases. PnhA hydrolyzes diadenosine tetra-, penta-, and hexaphosphates with a preference for diadenosine pentaphosphate, from which it forms ATP and ADP. PnhA requires a divalent metal cation, Mg(2+) or Mn(2+), and prefers an alkaline pH of 8 for optimal activity. A P. multocida strain that lacked a functional pnhA gene, ACP13, was constructed to further characterize the function of PnhA. The cellular size of ACP13 was found to be 60% less than that of wild-type P. multocida, but the growth rate of ACP13 and its sensitivity to heat shock conditions were similar to those of the wild type, and the wild-type cell size was restored in the presence of a functional pnhA gene. Wild-type and ACP13 strains were tested for virulence by using the chicken embryo lethality model, and ACP13 was found to be up to 1,000-fold less virulent than the wild-type strain. This is the first study to use an animal model in assessing the virulence of a bacterial strain that lacked a dinucleoside oligophosphate pyrophosphatase and suggests that the pyrophosphatase PnhA, catalyzing the hydrolysis of diadenosine pentaphosphates, may also play a role in facilitating P. multocida pathogenicity in the host.

Preliminary investigation of the use of high frequency ultrasound imaging in the chick embryo.

BACKGROUND: The purpose of this study was to investigate the feasibility of using high frequency ultrasound to study the chick embryo in a noninvasive and longitudinal fashion. METHODS: A total of 10 SPF White Leghorn chick embryos (GDs 11-17; Hamburger and Hamilton stage 37-43) were consecutively examined with a GE Logiq 400 ProSeries ultrasound unit using an 11-MHz small parts ultrasound probe. Access for ultrasound visualization of the embryos was accomplished by opening a 2-3-cm window either in the air cell over the blunt end of the egg or laterally over the embryo-dependent side of the egg. Warmed ultrasound coupling gel was used for imaging, and thermal regulation was maintained with infant heel warmers. The ultrasound images were recorded directly on digital video using a Sony TRV 900 DV camcorder. The images were directly converted to jpeg and mjeg2 files for further analysis. RESULTS: Effective visualization of each embryo was possible on each day of the study period. The embryos were best visualized through the opening made in the air cell at the blunt end of the egg. The extent of the anatomic survey of the chick embryo was dependent upon the position of the embryo in the egg relative to the opening in the air cell. Doppler color flow mapping studies were obtained of the embryonic and extraembryonic circulation. CONCLUSIONS: This preliminary investigation clearly shows the feasibility of high frequency ultrasound imaging to study chick embryo development in a longitudinal and noninvasive fashion. Further studies are presently ongoing regarding earlier embryo development, as well as to determine the stability and dynamics of the methodology.

Detection of Escherichia coli O157:H7 bacteria by a combination of immunofluorescent staining and capillary electrophoresis.

As the number of incidents of bacterial infections continues to rise around the globe, simpler, faster, and more sensitive diagnostic techniques are required to improve the safety of the food supply and to screen for potential bacterial infections in humans. We present here direct and indirect approaches for the detection of bacteria, which are based upon a combination of immunofluorescent staining and capillary electrophoresis. In the direct approach, Escherichia coli O157:H7 bacteria stained with fluorescein-tagged specific antibodies are detected by CE, while in the indirect approach fluorescein-tagged specific antibodies to E. coli are first captured by E. coli O157:H7 bacteria and then released and detected by CE. We have identified suitable bacteria staining and CE protocols, which involved a 10 mM Tris-borate-EDTA (TBE) buffer, 0.25 micro g antibody/1 million bacteria, and capillaries dynamically coated with poly-N-hydroxyethylacrylamide (polyDuramide). We have also successfully detected the presence of E. coli O157:H7 in contaminated meat. The total time required for analysis was 6-8 h, which is less than that realized in most commercial assays presently available.