Molecular investigation of a listeriosis outbreak in goats caused by an unusual strain of Listeria monocytogenes.

During a 16-month period, 10 goats with listeriosis were identified in 2 herds that shared 3 bucks, including 1 that died of listeriosis. Using DNA fingerprinting, we determined that a single genetically unique Listeria monocytogenes strain had infected all goats from which isolates were available. All isolates were unable to metabolize rhamnose (rhamnose-negative), whereas as a species, L monocytogenes is considered to have a rhamnose-positive phenotype. Therefore, these isolates would have been characterized as a species other than L monocytogenes if any of a variety of commercial bacterial identification kits had been used for speciation. Silage was not fed to either herd, and L monocytogenes was not isolated from vaginal or rectal swab specimens obtained from healthy goats or from samples of feed. Because the 3 bucks were the only common elements between the 2 herds, our results suggest a venereal route of transmission for listeriosis.

Urinary 8-hydroxydeoxyguanosine in infants and children.

Several diseases of prematurity are thought to be related to oxidative injury and many of the available markers are unsatisfactory. An assay was developed using HPLC with electrochemical detection for the quantitation of urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a proposed indicator for oxygen-derived free radical injury to DNA in preterm infants. A median value of 3.79 micromol/mol creatinine was obtained for normal children (2-15 years old, n=14). Urinary 8-OHdG excretion in neonates ranged from 0-99 micromol/mol creatinine. There were no gestation or birthweight related differences in urinary 8-OHdG, and no correlation with urinary malondialdehyde. Mean 8-OHdG excretion increased with postnatal age (r=0.80, p < 0.0001, n=15), mirroring the growth velocity curve. These changes could also be due to changes in the activity of the enzyme responsible for 8-OHdG excision. Urinary 8-OHdG levels are unlikely to accurately reflect oxygen derived free radical activity given the strength of the relationship with growth.

Picenadol in a large multicenter dental pain study.

STUDY OBJECTIVE: To estimate the analgesic dose of picenadol hydrochloride equal to codeine 60 mg in a dental pain model. DESIGN: Randomized, double-blind, parallel, dose-response study. SETTING: Four university-based dental clinics. PATIENTS: Four hundred eight adult patients with moderate or severe pain after extraction of one or more impacted molar teeth plus bone removal. INTERVENTIONS: Patients received orally administered single doses of picenadol 15 and 30 mg, codeine phosphate 30 and 90 mg, or placebo. METHODS: Single oral doses of picenadol 15 and 30 mg, an opioid agonist-antagonist, were compared with codeine 30 and 90 mg and placebo in 408 patients with moderate or severe pain from third molar extraction in a randomized, double-blind, parallel study. Assessments were performed for pain intensity, pain relief, and adverse events for up to 6 hours after drug administration. MAIN RESULTS: Picenadol 30 mg and codeine 90 mg were more effective than placebo based on sum of pain intensity differences, total pain relief, peak pain relief, and duration of analgesia (p < 0.05). Compared with placebo, the frequency of adverse events was highest for patients receiving codeine 90 mg (p < 0.05). No patients discontinued due to adverse events, and all such events resolved spontaneously. CONCLUSIONS: Picenadol 22 mg was estimated to be equianalgesic to codeine 60 mg, and picenadol 30 mg was safe in this dental pain model.

Plasma hypoxanthine: a marker for hypoxic-ischaemic induced periventricular leucomalacia?

Cerebral ischaemia of the immature brain may result in cavitating periventricular leucomalacia (PVL), an important association of cerebral palsy. Hypoxanthine measured by high performance liquid chromatography was used as a marker of peripartum hypoxia and ischaemia in 116 infants at risk of PVL. PVL was detected by ultrasound. The 81 infants who were unaffected had median (range) gestation of 30 weeks (24-32), weight of 1336 g (724-3790), and a plasma hypoxanthine concentration of 7.8 mumol/l (2.4-48.9). The seven infants who had cavitating PVL had a median gestation of 28 weeks (26-30), weight of 1165 g (682-1860), and a hypoxanthine concentration of 31.9 mumol/l (7.1-149). Cavitating PVL was significantly dependent only on hypoxanthine when controlling for the effects of weight and gestation. This suggests that peripartum hypoxia-ischaemia may be one of the aetiological factors in cavitating PVL. Oxidation of hypoxanthine during reperfusion generates free radicals which may contribute to the tissue destruction of PVL. The association of hypoxia-ischaemia with PVL suggests that PVL may be modified by reducing free radical activity.

Enhanced in vitro growth of murine fibroblast cells and preimplantation embryos cultured in medium supplemented with an amphipathic peptide.

Preliminary studies on the proliferative effects of lytic peptides were carried out using NIH 3T3 murine fibroblast cells and human lymphocytes. Cells were cultured in various concentrations of three different amphipathic peptides (SB-37, Shiva-1, and Vishnu), and enhanced proliferation was determined by uptake of 3H-thymidine with treated cells compared with control cultures. Enhanced proliferation of 3T3 cells was observed in cultures containing 50 microM or less SB-37. The primary study consisted of 263 four-cell- to eight-cell-stage mouse embryos from naturally bred mice and incubated in Whitten's medium containing 0.2, 1, or 10 microM of the amino terminus of an amphipathic cecropin B analog (Vishnu) or in Whitten's medium alone. Embryos were cultured to the hatched blastocyst stage, and effect of treatment was determined by the rate of growth to that stage of development. Statistical analysis revealed that culture in all three levels of Vishnu significantly accelerated in vitro growth of these stages of preimplantation embryos compared with controls. These results indicate that Vishnu promotes increased cleavage rates of embryos in vitro. A growth factor receptor clustering mechanism of action is proposed. This peptide may have some potential as an embryo culture medium additive to enhance in vitro growth rate.

Experimental infection of goat fetuses in utero with a stable, rough mutant of Brucella abortus.

Fetuses of goats in their last trimester of pregnancy were experimentally infected with Brucella abortus strain RB51, a stable rough mutant deficient in the perosamine O-chain content of its lipopolysaccharide. RB51 maintained its rough phenotype in vivo and did not induce abortion. Infection with RB51 resulted in the production of significant levels of IgG type antibodies specific for B abortus cellular antigens distinct from the perosamine O-chain. These findings suggest that strain RB51 will be useful in the pregnant goat for studying the role of brucella antigens other than the lipopolysaccharide O-chain in the immune response to brucellosis.

In vitro cytocidal effect of lytic peptides on several transformed mammalian cell lines.

Several types of transformed mammalian cells, derived from established cell lines, were found to be lysed in vitro by three novel lytic peptides (SB-37, SB-37*, and Shiva-1). This is in contrast with the behavior of normal cells, where the observed lytic activity of the peptides is greatly reduced. Based on experiments utilizing compounds which disrupt the cytoskeleton (colchicine and cytochalasin-D), it is surmised that alterations in the cytoskeleton of transformed cells increase their sensitivity to the cytolytic activity exerted by the peptides, primarily by causing a loss of osmotic integrity. Thus, a stable and regenerative cytoskeletal system, as that possessed by normal cells, would seem requisite to withstanding the lytic effects of the peptides.

In vivo activation of equine eosinophils and neutrophils by experimental Strongylus vulgaris infections.

Eosinophils and neutrophils from ponies with Strongylus vulgaris-induced eosinophilia (eosinophilic ponies; activated eosinophils and neutrophils) were assayed in vitro for chemotactic and chemokinetic responses to zymosan-activated serum (ZAS) using the filter system in Boyden chambers, for Fc and complement (C) receptors using the EA and EAC-rosette assays, respectively, and for phagocytic and bactericidal activities using opsonized Escherichia coli and the acridine orange method. The responses of activated eosinophils and neutrophils in the above assays were compared with those of eosinophils and neutrophils from S. vulgaris-naive ponies without eosinophilia (noneosinophilic ponies; nonactivated eosinophils and neutrophils). Differences in cell density following centrifugation in a continuous Percoll gradient were used to further characterize the heterogeneity of activated eosinophils and neutrophils. Activated and nonactivated eosinophils demonstrated similar chemotactic responses to ZAS while activated and nonactivated neutrophils demonstrated similar chemokinetic responses to ZAS. A higher percentage of activated eosinophils and neutrophils expressed Fc and C receptors compared with nonactivated cells (P less than 0.05). Generally, higher percentages of eosinophils and neutrophils expressed C than Fc receptors. However, the percentage of neutrophils with both receptors was higher than that of eosinophils. Phagocytosis and killing of E. coli by either type of eosinophil were not consistently observed. Both activated and nonactivated neutrophils phagocytized E. coli and significant differences between the two cell types were not observed. The bacterial activity, however, of activated neutrophils was significantly greater than that obtained using nonactivated neutrophils (P less than 0.05). Activated eosinophils and neutrophils were both separated into two distinct fractions based on differences in cell densities. A higher percentage of band 2 eosinophils (density of 1.106) expressed C receptors than did band 1 eosinophils (density of 1.049) (P less than 0.05). A higher percentage of band 1 neutrophils (density of 1.072) expressed both Fc and C receptors and these neutrophils were more phagocytic and bactericidal than were band 2 neutrophils (density of 1.082) (P less than 0.05). These data suggest that equine eosinophils and neutrophils are activated by chronic S. vulgaris infections.

In vitro cytocidal effect of novel lytic peptides on Plasmodium falciparum and Trypanosoma cruzi.

Plasmodium falciparum and Trypanosoma cruzi were killed by two novel lytic peptides (SB-37 and Shiva-1) in vitro. Human erythrocytes infected with P. falciparum, and Vero cells infected with T. cruzi, were exposed to these peptides. The result, in both cases, was a significant decrease in the level of parasite infection. Furthermore, the peptides had a marked cytocidal effect on trypomastigote stages of T. cruzi in media, whereas host eukaryotic cells were unaffected by the treatments. In view of the worldwide prevalence of these protozoan diseases and the lack of completely suitable treatments, lytic peptides may provide new and unique chemotherapeutic agents for the treatment of these infections.

The granulomatous inflammatory response in jirds, Meriones unguiculatus, to Brugia pahangi: an ultrastructural and histochemical comparison of the reaction in the lymphatics and peritoneal cavity.

A granulomatous inflammatory response develops in jirds with lymphatic or intraperitoneal infections of Brugia pahangi. Light, histochemical, and ultrastructural microscopy were used for comparative studies of the reactions in these 2 locations. The reactions observed were categorized into 3 types: (1) an initial response in which lymphocytes, monocytes, macrophages, and eosinophils were present; (2) an intermediate one which consisted of macrophages, epithelioid cells, lymphocytes, eosinophils, collagen, and mesothelial/endothelial cells with central areas of necrosis; and (3) a terminal reaction consisting of degenerating, necrotic cells. Microfilariae and adult worms were associated with these reactions. Macrophages were the predominant cell type in the lesion and were often found attached to the surface of the parasite. The inflammatory responses to B. pahangi in the lymphatics and in the peritoneal cavity appear to be similar, and thus, the peritoneal cavity may be useful in studying specific cell-parasite interactions to further define the pathogenesis of filarial disease.

Cellular and humoral responses of Brucella abortus-infected bovine fetuses.

Fifty-nine bovine fetuses naturally and experimentally infected with Brucella abortus were studied. Lymphoid hyperplasia in multiple lymph nodes, lymphoid depletion in the thymic cortex, adrenal cortical hyperplasia, and disseminated inflammatory foci composed mainly of large mononuclear leukocytes were present in infected fetuses. Histopathologic changes in naturally infected fetuses were indistinguishable from those infected fetuses inoculated in utero. Fetuses inoculated with 1.0 X 10(3) to 1.0 X 10(5) colony-forming units of strain 2308 B abortus were aborted on postinoculation day (PID) 7 to 19. Fetuses obtained by PID 9 and 10 had increased immunoglobulin concentrations and antibody. Increased cortisol values were present in fetuses obtained as early as PID 6. The initial fetal inflammatory response was composed of large mononuclear leukocytes. In fetuses obtained by PID 9 to 10, moderate numbers of neutrophils mixed with mononuclear leukocytes were present in the inflammatory foci. This shift in the initial inflammatory reaction coincided with the appearance of agglutinating antibody.

Activation of jird (Meriones unguiculatus) macrophages by the filarial parasite Brugia pahangi.

Peritoneal macrophages from Mongolian jirds (Meriones unguiculatus) with either lymphatic or intraperitoneal infections of Brugia pahangi were studied to determine the effects of infection on macrophage function and morphology. Macrophages were collected at 40, 90, 140, and 200 days after inoculation of infective third-stage larvae and assayed for phagocytic and bactericidal activity by the acridine orange method and for morphological changes by light and electron microscopy. Significant increases in phagocytic and microbicidal activity (P less than or equal to 0.01) were observed in peritoneal macrophages collected from jirds with intraperitoneal infections when compared with peritoneal macrophages from jirds with lymphatic infections and resident peritoneal macrophages from normal, noninfected jirds. Morphological changes in peritoneal macrophages from jirds with intraperitoneal infections were similar to those found in thioglycolate-elicited macrophage populations. Granuloma formation was also observed in the peritoneal cavities of intraperitoneally infected jirds. The peritoneal cavity may serve as a model to study cell-worm interactions in filarial nematode infections.

Equine infectious anemia virus: development of a simple reproducible method for titrating infectivity of the cell-adapted strain.

An equine dermal cell line between the 14th and 30th subpassages was used to develop a reproducible method of titrating the infectivity of the cell-adapted strain of equine infectious anemia virus (EIAV). Cells inoculated with EIAV were subcultured or fed once each week and were monitored for the production of P26 antigen of EIAV in supernatant fluids. Ultrathin sections were prepared once each week and were examined for the detection of budding virus-like particles (VLP). The VLP could not be detected in the infected cells subcultured once each week. When the medium was changed once week and when the cells not transferred, budding VLP were detected routinely after 3 to 4 weeks, Feeding of the infected cells once each week was used to establish the infectivity assay. The reproducibility of the assay on a frozen viral stock preparation was within an endpoint of one tenfold dilution.