Involvement of Pore Formation and Osmotic Lysis in the Rapid Killing of Gamma Interferon-Pretreated C166 Endothelial Cells by Rickettsia prowazekii.

Rickettsia prowazekii, the bacterial cause of epidemic typhus in humans, proliferates mainly within the microvascular endothelial cells. Previous studies have shown that murine macrophage-like RAW264.7 cells are rapidly damaged if they are pretreated with gamma interferon (IFN-γ) and then infected with R. prowazekii. In the present study, the effects of IFN-γ and R. prowazekii on murine C166 endothelial cells were evaluated. In the IFN-γ-pretreated R. prowazekii-infected endothelial cell cultures, evidence of cell damage was observed within several hours after addition of the rickettsiae. Considerable numbers of the cells became permeable to trypan blue dye and ethidium bromide, and substantial amounts of lactate dehydrogenase (LDH) were released from the cells. Such evidence of cellular injury was not observed in the untreated infected cultures or in any of the mock-infected cultures. Polyethylene glycols (PEGs) of different nominal average molecular weights were used to assess the possible involvement of pore formation and osmotic lysis in this cellular injury. PEG 8000 dramatically suppressed LDH release, PEG 4000 partially inhibited it, and PEGs 2000 and 1450 had no effect. Despite its inhibition of LDH release, PEG 8000 did not prevent the staining of the IFN-γ-pretreated infected endothelial cells by ethidium bromide. These findings suggest that the observed cellular injury involves the formation of pores in the endothelial cell membranes, followed by osmotic lysis of the cells.

Detection of nitric oxide production in cell cultures by luciferin-luciferase chemiluminescence.

A chemiluminescent method is proposed for quantitation of NO generation in cell cultures. The method is based on activation of soluble guanylyl cyclase by NO. The product of the guanylyl cyclase reaction, pyrophosphate, is converted to ATP by ATP sulfurylase and ATP is detected in a luciferin-luciferase system. The method has been applied to the measurement of NO generated by activated murine macrophages (RAW 264.7) and bovine aortic endothelial cells. For macrophages activated by lipopolysaccharide and γ-interferon, the rate of NO production is about 100 amol/(cell·min). The rate was confirmed by the measurements of nitrite, the product of NO oxidation. For endothelial cells, the basal rate of NO generation is 5 amol/(cell·min); the rate approximately doubles upon activation by bradykinin, Ca(2+) ionophore A23187 or mechanical stress. For both types of cells the measured rate of NO generation is strongly affected by inhibitors of NO synthase. The sensitivity of the method is about 50 pM/min, allowing the registration of NO generated by 10(2)-10(4) cells. The enzyme-linked chemiluminescent method is two orders of magnitude more sensitive than fluorescent detection using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM).

Serologic survey of wild turkeys (Meleagris gallopavo) and evidence of exposure to avian encephalomyelitis virus in Georgia and Florida, USA.

Wild Turkeys (Meleagris gallopavo) are susceptible to many of the same diseases as domestic turkeys. Before 2005, most Wild Turkeys in southern Georgia, US, had little or no exposure to commercial poultry operations. As part of a pathogen survey examining the effects of commercial poultry on Wild Turkeys, samples were collected from Wild Turkeys from March 2005 through May 2008. The turkeys were collected from 13 counties in southern Georgia and Madison County, Florida, and tested for antibodies to various pathogens of poultry. Three (13%) of the turkeys were positive for antibodies to Salmonella. Thirteen turkeys (54%) were positive for Newcastle disease virus antibodies, and 15 turkeys (63%) were positive for antibodies to reticuloendotheliosis virus. One turkey (4%) from Madison County was positive for avian encephalomyelitis virus antibodies.

Salmonella from gopher tortoises (Gopherus polyphemus) in south Georgia.

From 2002 to 2006, gopher tortoises (Gopherus polyphemus) were collected at Moody Air Force Base, Lowndes/Lanier counties, Georgia, USA, and opportunistically surveyed for the presence of Salmonella species. Four of 155 (2.6%) cloacal swabs collected from 80 tortoises were positive for the presence of Salmonella enterica, and the following serovars were identified: Give, Hartford, Javiana, and Luciana. Female tortoises (5%) were infected at a rate similar to male tortoises (5%). All isolates were obtained from adult tortoises (n = 73); subadults (n = 7) were all negative. Each isolated serovar is a potential human pathogen, suggesting appropriate precautions should be emphasized when handling these animals.