Comparative nitric oxide production by LPS-stimulated monocyte-derived macrophages from Ovis canadensis and Ovis aries.

Bighorn sheep are more susceptible to respiratory infection by Mannheimia haemolytica than are domestic sheep. In response to bacterial challenge, macrophages produce a number of molecules that play key roles in the inflammatory response, including highly reactive nitrogen intermediates such as nitric oxide (NO). Supernatants from monocyte-derived macrophages cultured with M. haemolytica LPS were assayed for nitric oxide activity via measurement of the NO metabolite, nitrite. In response to LPS stimulation, bighorn sheep macrophages secreted significantly higher levels of NO compared to levels for non-stimulated macrophages. In contrast, levels of NO produced by domestic sheep macrophages in response to M. haemolytica LPS did not differ from levels detected in non-stimulated cell cultures. Nitrite levels detected in supernatants of LPS-stimulated bighorn macrophage cultures treated with an inducible nitric oxide synthase (INOS) inhibitor, N(G)-monomethyl-L-arginine, were similar to that observed in non-stimulated cultures indicating a role for the iNOS pathway.

Evaluation of ewe vaccination as a tool for increasing bighorn lamb survival following pasteurellosis epizootics.

We conducted field and laboratory experiments to evaluate whether treating pregnant bighorn ewes with a combination of an experimental Pasteurella trehalosi and Mannheimia haemolytica (formerly P. haemolytica) vaccine and a commercially-available bovine P. multocida and M. haemolytica vaccine would increase lamb survival following a pneumonia epidemic. Three free-ranging bighorn herds affected by pasteurellosis outbreaks between November 1995 and June 1996 were included in the field experiment. Post-epidemic lamb survival was low in all three herds in 1996, with November lamb:ewe ratios of < or = 8:100. In March 1997, thirty-six ewes (12/herd) were captured and radiocollared. Half of the ewes captured in each herd were randomly selected to receive both vaccines; the other half were injected with 0.9% saline solution as controls. Lambs born to radiocollared ewes were observed two or more times per week and were considered to have survived if they were alive in October 1997, about 6 mo after birth. Lamb survival differed among herds (range 22% to 100%), and survival of lambs born to vaccinated ewes was lower (P = 0.08) than survival of lambs born to unvaccinated ewes. Bronchopneumonia (pasteurellosis) was the dominant cause of mortality among lambs examined. We concurrently evaluated vaccine effects on survival of lambs born to seven captive ewes removed from the wild during the 1995-96 epidemic. Antibody titers were high in captive ewes prior to vaccination, and vaccines failed to enhance antibody titers in treated captive ewes. None of the captive-born lambs survived. These data suggest that, using existing technology, vaccinating bighorn ewes following pneumonia epidemics has little chance of increasing neonatal survival and population recovery.

Immunogenicity of a heptavalent pneumococcal conjugate vaccine in Apache and Navajo Indian, Alaska native, and non-native American children aged <2 years.

High rates of invasive pneumococcal disease have been described among infants living in various Native American communities. In this study, we evaluated the immunogenicity of a 7-valent pneumococcal vaccine consisting of serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F covalently linked to the outer membrane protein complex of Neisseria meningitidis in Apache and Navajo Indian, Alaska Native, and non-Native American children. The vaccine was administered at ages 2, 4, and 6 months; a booster dose was given at age 15 months. Levels of serotype-specific immunoglobulin G (IgG) were measured by a standardized enzyme-linked immunosorbent assay. The responses after 3 primary doses of vaccine were similar in all 3 groups of children, except for those to serotypes 14 and 23F. One month after the booster dose, geometric mean concentrations (GMCs) of serotype-specific IgG antibodies increased significantly in all 3 groups of children, compared with GMCs of IgG antibodies to pneumococcal serotypes before the booster dose.

Serotype distribution and antimicrobial resistance patterns of invasive isolates of Streptococcus pneumoniae: Alaska, 1991-1998.

From January 1991 through December 1998, a total of 1046 pneumococcal isolates were received from 23 laboratories participating in the statewide surveillance system. Of these, 1037 were recovered from normally sterile sites (blood and cerebrospinal and pleural fluid) and were available for serotyping and susceptibility testing. Ninety-two percent of these isolates were serotypes represented in the 23-valent pneumococcal polysaccharide vaccine. Serotypes in the 7-valent pneumococcal conjugate vaccine (4, 6B, 9V, 14, 18C, 19F, and 23F) were recovered from 72% of Alaska Natives and 84% of non-Native children <5 years old with invasive disease. Statewide, 7.3% and 3.2% of isolates had intermediate and high levels of resistance to penicillin, respectively; 9.2% were resistant to erythromycin (minimal inhibitory concentration, >/=1 microg/mL) and 19% to trimethoprim/sulfamethoxazole (minimal inhibitory concentration, >/=4/76 microg/mL). Twelve percent of invasive isolates were resistant to >/=2 classes of antibiotics; of these, serotype 6B accounted for 33%, and 63% were recovered from children <5 years old.

Characterization of a multidrug-resistant clone of invasive Streptococcus pneumoniae serotype 6B in Alaska by using pulsed-field gel electrophoresis and PspA serotyping.

Antimicrobial susceptibility, pneumococcal surface protein A (PspA) serotyping, and pulsed-field gel electrophoresis (PFGE) were used to evaluate clonal relatedness among 66 invasive isolates of Streptococcus pneumoniae serotype 6B collected during 1982-1996 from patients in Alaska. Thirty-seven (56%) of the isolates had penicillin minimal inhibitory concentration values >/=0.125 microgram/mL and were resistant to at least 1 other antibiotic. Fourteen PspA serotypes were observed; PspA 16 was the most common (35%). Forty-five (68%) of the 66 isolates shared common and highly related PFGE patterns using 3 enzymes. Twenty-six (58%) of the isolates with common PFGE patterns were from Native Alaskan children

Immunologic responses of domestic and bighorn sheep to a multivalent Pasteurella haemolytica vaccine.

The efficacy of a Pasteurella haemolytica vaccine (serotypes A1, A2, and T10) to induce humoral antibodies and alter colonization of the upper respiratory tract by related P. haemolytica spp. strains was evaluated in 10 bighorn (Ovis canadensis canadensis) and 10 domestic (Ovis aries) sheep. Sheep of each species were divided into five pairs based on age and history of respiratory disease. One sheep in each pair was vaccinated twice 2 wk apart with 2 ml of vaccine (VAC group) and the remaining animals (NV group) were injected with 2 ml of sterile saline. Mild, transient lameness was the only observed adverse effect. Blood sera from the sheep were tested for agglutinating antibodies against whole cells of A1, A2, and T10 and for leukotoxin neutralizing antibodies. Antibody titers were expressed as the reciprocal log2 of the highest reactive dilutions. Domestic sheep > 1-yr-old and two bighorn sheep with a history of A1 infection had higher titers throughout the study against A1 cells than domestic sheep < 1-yr-old and bighorns without a history of A1 infection. Both domestic and bighorn sheep had log2 titers of 8 to 12 against A2 cells and 6 to 12 against T10 cells during this time. Bighorn sheep in the VAC group had 2 to 32 fold titer increases for A1 cells by 2 wk post-vaccination (PV) compared to 0 to 2 fold increases in VAC domestic sheep. Two to 16 and 0 to 8 fold increases in antibodies titers to A2 and T10 cells, respectively, were detected in sera of both VAC groups. Sera of bighorn sheep with a history of respiratory disease and all domestic sheep had log2 leukotoxin neutralizing antibody titers of 4 to 14 in contrast to < or = 2 in sera of bighorn sheep without a history of respiratory disease. Neutralizing antibody titers of two bighorns without a history of respiratory disease in the VAC group increased from log2 0 to 5 in one and from 0 to 9 in the other 2 wk PV. Antibody increases in these animals were no longer evident at 16 wk PV while titers of animals with histories of disease remained relatively stable. The types and numbers of Pasteurella spp. isolated from nasal and pharyngeal swabs varied throughout the study without conclusive evidence of suppression of colonization. Although the animals were not experimentally challenged to determine the efficacy of the vaccine, one VAC and one NV bighorn sheep died following introduction of an A2 P. haemolytica strain when leukotoxin neutralizing antibodies had returned to pre-vaccination levels. This vaccine appeared to be safe for use in bighorn sheep and stimulated moderate but transient increases in antibody levels which should provide some protection against naturally occurring disease. A vaccine which would induce production of high and maintained antibodies against multiple strains of P. haemolytica would be valuable for use in bighorn sheep maintained in captivity or when captured for relocation.

Molecular analysis by pulsed-field gel electrophoresis and antibiogram of Streptococcus pneumoniae serotype 6B isolates from selected areas within the United States.

Fifty-eight clinical isolates of Streptococcus pneumoniae serotype 6B, including 16 from Alaska, 14 from Arizona, 11 from Washington, and 17 from seven additional states, were analyzed. The antibiograms of these isolates were assigned to 10 antibiotic profiles based on their susceptibilities to penicillin, erythromycin, tetracycline, and trimethoprim-sulfamethoxazole. Thirty-two (55%) of these isolates were penicillin nonsusceptible, while 21 (36%) were intermediate or resistant to three or more antibiotics. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by pulsed-field gel electrophoresis (PFGE). The ApaI and SmaI PFGE patterns were combined, and 13 of the 16 Alaskan isolates showed indistinguishable PFGE patterns. One other isolate exhibited highly related ApaI and SmaI PFGE patterns, differing by only one band after restriction with ApaI. Among the 14 isolates from Arizona, 1 was indistinguishable from the predominant ApaI and SmaI PFGE patterns seen in the Alaskan isolates; 5 others were highly related (+/-1 band after cutting with either enzyme) to the Alaskan isolates, suggesting a common ancestral origin. Of the remaining eight isolates, six additional ApaI plus SmaI PFGE patterns were observed. The 28 isolates from the various contiguous states had 22 ApaI plus SmaI PFGE patterns. No correlations were found between specific PFGE patterns, antibiograms, dates of isolation, or geography. The serotype 6B isolates across the contiguous United States were genetically diverse, while the 6B isolates from Alaska appeared to be much less diverse.

Complex regulatory region mediating tailless expression in early embryonic patterning and brain development.

tailless encodes a transcription factor expressed in multiple domains in the developing embryo. Early and transient expression at the posterior pole is required to establish a domain from which the eighth abdominal segment, telson and posterior gut arise. Just a few nuclear cycles later, a brain-specific domain is initiated at the anterior; expression in this domain is maintained with complex modulations throughout embryogenesis. Expression of tailless in this domain is required to establish the most anterior region of the brain. To understand the function and regulation of these different domains of expression, we provide a detailed description of tailless expression in brain neuroblasts and show that this expression is not detectably regulated by the head gap genes buttonhead or orthodenticle, by the proneural gene lethal of scute or by tailless itself. We show that approximately 6 kb of sequenced upstream regulatory DNA can drive lacZ expression in a pattern that mimics the full tailless embryonic expression pattern. Within this sequence we identify multiple modules responsible for different aspects of the tailless pattern. In addition to identifying additional torso response elements that mediate early blastoderm polar expression, we show that the complex brain expression pattern is driven by a combination of modules; thus expression at a low level throughout the brain and at a high level in the dorsal medial portion of the brain and in the optic lobe, as well as neuroblast-specific repression are mediated by different DNA regions.

The torso response element binds GAGA and NTF-1/Elf-1, and regulates tailless by relief of repression.

Modulation of transcription factor activity leading to changes in cell behavior (e.g., differentiation versus proliferation) is one of the critical outcomes of receptor tyrosine kinase (RTK) stimulation. In the early Drosophila embryo, activation of the torso (tor) RTK at the poles of the embryo activates a phosphorylation cascade that leads to the spatially specific transcription of the tailless (tll) gene. Our analysis of the tor response element (tor-RE) in the tll promoter indicates that the key activity modulated by the tor RTK pathway is a repressor present throughout the embryo. We have mapped the tor-RE to an 11-bp sequence; using this sequence as the basis for protein purification, we have determined that the proteins GAGA and NTF-1 (also known as Elf-1, product of the grainyhead gene) bind to the tor-RE. We demonstrate that NTF-1 can be phosphorylated by MAPK (mitogen-activated protein kinase), and that tll expression is expanded in embryos lacking maternal NTF-1 activity; these results make NTF-1 a likely target for modulation by the tor RTK pathway in vivo. The data presented here support a model in which activation of the tor RTK at the poles of the embryos leads to inactivation of the repressor and therefore, to transcriptional activation (by activators present throughout the embryo) of the tll gene at the poles of the embryo.

Measurement of pneumococcal capsular polysaccharide serotype-specific immunoglobulin G in human serum, a method for assigning weight-based units to proposed reference sera.

A direct method for measuring serotype-specific, class-specific antibody in proposed human reference sera is described. The assay uses a 125I-labeled, isotopically pure immunoglobulin G (IgG) as a primary standard in an antigen-antibody solid-phase enzyme immunoassay. From the measurement of specific radioactivity bound to the absorbed antigen, serotype-specific IgG concentrations and optical density values can be directly related to optical density and serotype-specific IgG values for the reference serum. We used this method to provisionally assign IgG concentrations in a pneumococcal reference serum to serotypes 1, 3, 6A, 12F, 14 and 23F. This assay was found to be reproducible; the coefficient of variation for duplicates was within 5%, and the day-to-day coefficient of variation was from 3 to 18% for all six serotypes. The assay provides a general method for standardizing human reference serum tools with respect to concentration of antigen-specific IgM-, IgA-, and IgG-subclass antibodies.

Evaluation of polymerase chain reaction for diagnosis of pneumococcal pneumonia.

To test the ability of the polymerase chain reaction (PCR) to detect Streptococcus pneumoniae in blood, we generated two sets of nested primers. The first defined 559-bp and 649-bp regions of the pneumolysin gene, and the second defined 445-bp and 553-bp regions of the autolysin gene. These nucleotide segments were detected in DNAs from isolates of all 20 pneumococcal serotypes tested, but they were not detected when used to test DNAs from 41 isolates of nonpneumococcal bacteria and fungi. The sensitivity was evaluated by using purified pneumococcal DNA. We were able to detect 10 fg of S. pneumoniae DNA, or 4.3 genome equivalents. Blood samples were obtained from 16 patients with culture-proven pneumococcal bacteremia and were subjected to PCR analysis. Of eight buffy coat fractions tested, six showed reactivity in the PCR with the pneumolysin primers, and five of the eight produced the expected products when tested with the autolysin primers (sensitivities, 75 and 63%, respectively). Of the eight whole-blood specimens tested, only three produced the expected products with either set of primers. Additionally, we tested 14 samples from patients with bacteremia that were culture positive for nonpneumococcal bacterial species, and 13 were negative (specificity, 93%). This combination of sensitivity and specificity may make detection of S. pneumoniae in blood by PCR in comparison with that by blood culture a very promising alternative for a means of definitive diagnosis.