Thomas J. Doyle

Pathogenesis of Infection by Clinical and Environmental Strains of Vibrio vulnificus in Iron-Dextran-Treated Mice

ABSTRACT Vibrio vulnificus is an opportunistic pathogen that contaminates oysters harvested from the Gulf of Mexico. In humans with compromising conditions, especially excess levels of iron in plasma and tissues, consumption of contaminated seafood or exposure of wounds to contaminated water can lead to systemic infection and disfiguring skin infection with extremely high mortality. V. vulnificus-associated diseases are noted for the rapid replication of the bacteria in host tissues, with extensive tissue damage. In this study we examined the virulence attributes of three virulent clinical strains and three attenuated oyster or seawater isolates in mouse models of systemic disease. All six V. vulnificus strains caused identical skin lesions in subcutaneously (s.c.) inoculated iron dextran-treated mice in terms of numbers of recovered CFU and histopathology; however, the inocula required for identical frequency and magnitude of infection were at least 350-fold higher for the environmental strains. At lethal doses, all strains caused s.c. skin lesions with extensive edema, necrosis of proximate host cells, vasodilation, and as many as 108CFU/g, especially in perivascular regions. These data suggest that the differences between these clinical and environmental strains may be related to growth in the host or susceptibility to host defenses. In non-iron dextran-treated mice, strains required 105-fold-higher inocula to cause an identical disease process as with iron dextran treatment. These results demonstrate that s.c. inoculation of iron dextran-treated mice is a useful model for studying systemic disease caused by V. vulnificus.

Virulence Plasmid-Borne spvB and spvC Genes Can Replace the 90-Kilobase Plasmid in Conferring Virulence to Salmonella enterica Serovar Typhimurium in Subcutaneously Inoculated Mice

In a mouse model of systemic infection, the spv genes carried on the Salmonella enterica serovar Typhimurium virulence plasmid increase the replication rate of salmonellae in host cells of the reticuloendothelial system, most likely within macrophages. A nonpolar deletion in the spvB gene greatly decreased virulence but could not be complemented by spvB alone. However, a low-copy-number plasmid expressing spvBC from a constitutive lacUV5 promoter did complement the spvB deletion. By examining a series of spv mutations and cloned spv sequences, we deduced that spvB and spvC could be sufficient to confer plasmid-mediated virulence to S. enterica serovar Typhimurium. The spvBC-bearing plasmid was capable of replacing all of the spv genes, as well as the entire virulence plasmid, of serovar Typhimurium for causing systemic infection in BALB/c mice after subcutaneous, but not oral, inoculation. A point mutation in the spvBC plasmid preventing translation but not transcription of spvC eliminated the ability of the plasmid to confer virulence. Therefore, it appears that both spvB and spvC encode the principal effector factors for Spv- and plasmid-mediated virulence of serovar Typhimurium.

Comparative analysis of in vivo and in vitro AAV vector transduction in the neonatal mouse retina: effects of serotype and site of administration.

The specificity of retinal cells transduced by AAV serotype 1, 2 or 5 vectors was determined in vivo versus in vitro in the normal P7 mouse in order to develop a rapid and accurate way to anticipate the behavior of AAV vectors in the retina. In vivo results confirm that AAV1 transduces retinal pigment epithelial cells, while AAV2 and AAV5 transduce both RPE and photoreceptor cells by subretinal injection. AAV2 was the only serotype to efficiently transduce inner retinal cells by intravitreal injection. Parallel analysis employing in vitro retinal organ culture showed qualitatively similar AAV-mediated GFP expression as seen in vivo suggesting that organ culture substitute is a useful method to screen new vector transduction patterns, particular in retinal cells in neonatal mice.

Exponential-phase expression of spvA of the Salmonella typhimurium virulence plasmid: induction in intracellular salts medium and intracellularly in mice and cultured mammalian cells.

The spv genes of Salmonella typhimurium and other non-typhoidal Salmonella serovars are essential for efficient systemic infection beyond the intestines in orally inoculated mice as a model for enteric fever. These virulence genes are not significantly expressed by salmonellae during exponential growth in L broth but are induced when the bacteria enter the stationary phase of growth. Using RNase protection analysis to directly measure spvA mRNA from the virulence plasmid of S. typhimurium, we found that spvA was maximally induced in an SpvR- and RpoS-dependent manner during exponential growth in intracellular Salts Medium, which mimics the intracellular environment of mammalian cells. A cloned spvA-lacZ operon fusion in S. typhimurium was induced intracellularly in periotoneal cells of mice, correlating in vivo intracellular gene expression with intracellular function of the spv genes in infected mice. spvA was also induced intracellularly in vitro within both Henle-407 intestinal epithelial cells and J774.A1 macrophage-like cells when the bacteria were replicating with exponential kinetics. Prevention of invasion of salmonellae with cytochalasin D inhibited spvA induction within tissue culture cells, indicating that salmonellae must be internalized for spvA to be induced. The spvA-lacZ fusion was not induced by salmonellae in extracellular fluid of the peritoneal cavity or in serum. Since induction of the spv genes occurs intracellularly during exponential growth of salmonellae, cessation of growth may not be the most relevant inducing signal for spv gene expression.

Growth of Staphylococcus aureus in Diprivan and Intralipid Implications on the Pathogenesis of Infections

BACKGROUND The incidence and severity of infections are increased when Intralipid or Diprivan are administered to patients. Intralipid promotes infection, presumably by inhibiting the reticuloendothelial system, thereby suppressing the hosts constitutive immunity, whereas Diprivan supposedly promotes infection by supporting bacterial growth and increasing the inoculating dose. This study considers whether bacterial replication alone in Intralipid and Diprivan adequately explains the increased risk of infection associated with these agents or whether other factors might also be involved. METHODS Staphylococcus aureus was cultured in 10% Intralipid or Diprivan at clinically relevant conditions or in Intralipid containing 0.005% (w/v) sodium EDTA, a current additive, to measure growth. To determine whether Intralipid affected infection, New Zealand white rabbits were injected intravenously with S. aureus with or without Intralipid. Twenty-four hours later, bacteria in lung, liver, spleen, and kidney tissues were enumerated. RESULTS S. aureus failed to grow in Diprivan or Intralipid containing 0.005% EDTA. Whereas S. aureus did replicate in plain Intralipid, growth was delayed until the bacteria conditioned the media. Once initiated, growth was slow at clinically relevant temperatures. The administration of Intralipid to rabbits significantly increased the recovery of staphylococci from the kidneys, P < 0.001, relative to the other tissues 24 h after an intravenous inoculation with S. aureus, compared with rabbits receiving S. aureus with no Intralipid. CONCLUSIONS These results suggest that Diprivan, and possibly Intralipid, represent poor media for the growth of S. aureus and may promote infection through mechanisms other than increased inoculum size.

Resistance and disease in Brugia malayi infection of ferrets following prior infection, injection of attenuated infective larvae and injections of larval extracts

A partial resistance expressed by a 53% to 78% reduction in lymphatic filariae from a challenge infection was induced in ferrets (Mustela putorius furo) by a prior infection and by injection of radiation attenuated infective larvae (L3) but not by injections of lyophilized microfilariae (mf) or L3. Equivalent acquired resistance was demonstrated with and without overt filarial disease. A prior infection resulted in peripheral lymphoedema in approximately one‐third of the amicrofilaraemic resistant ferrets following challenge infection and injection of attenuated larvae resulted in inflammatory responses characteristic of a hyper‐responsive syndrome in one‐half of the amicrofilaraemic ferrets. Injections of lyophilized mf inhibited microfilaraemia and promoted development of lymphostatic disease. A limited examination of immune responses and histopathology suggested that disease in partially resistant ferrets was associated with high TH2 dependent responses directed, at least in part, to mature filariae and to mf. Mechanisms of resistance were not identified.

Mechanism of Pathogenicity in Salmonella abortusovis

Salmonella abortusovis is a serotype of S. enterica and causes a contagious infection in sheep leading to bacteraemia and colonization of the placenta followed by abortion. After the infection, in the first pregnancy, a natural immunity is generally acquired by sheep. Frequent mortality in lambs has also been reported. The infection is widely spread to flocks in Europe, and due to the high costly damages to a sheep-based farming, and since antibiotic treatment is ineffective, prevention by vaccination should be encouraged. However most of the dead or living vaccines commercialized at present, give only partial protection, and their effectivenes is often related to specific geographic areas (Pardon et al, 1990). More efforts should be made for the development of a genetically engineered vaccine that would be a better candidate for reliable protection. Since no major phenotypic characters are suitable, the epidemiology based on the molecular features of the pathogenic strains could help to understand the routes of dissemination (Helmuth et al. 1985, Colombo et al. 1992).

Enhanced inflammation to Brugia malayi microfilariae in ferrets infected with Trichinella spiralis.

Intravenous (i.v.) injection ofmicrofilariae (mf) ofB. malayi in the ferret (Mustella putoriusfuro) has proven to be a convenient experimental method for study of the pathogenesis of occult filariasis. Following i.v. injection of mf, ferrets develop eosinophilia, immune clearance of circulating mf, and inflammatory responses to mf characteristic of tropical filarial eosinophilia or the Meyers-Kouwenaar (MK) syndrome of occult filariasis in man (Thompson et al., 1985, Experimental Parasitology 60: 181-194). Lesions visible on the surface of the liver, principally abscesses ofeosinophils measuring 1-2 mm in diameter, have been used to evaluate the inflammatory reactivity to mf. The number of lesions that develop following i.v. injection of mf is positively correlated with the level of peripheral blood eosinophils, but the relationship of lesion development to immune responses or hypersensitivity remains unclear (Thompson et al., 1985, loc. cit.). A preliminary observation indicated that Trichinella infection in ferrets induced eosinophilia and an enhanced inflammatory reaction to injected mf without evidence of cutaneous hypersensitivity to antigens ofmf. The objectives of the present study were to confirm the enhanced inflammatory reactivity to mf induced by Trichinella infection and to characterize the cellular and immune responses to mf in these ferrets which, it was considered, could help clarify mechanisms active in the pathogenesis of occult filariasis. The experimental procedures and immunologic assays have been described previously (Thompson et al., 1985, loc. cit.; Thompson et al., 1986, Zeitschrift fur Parasitenkunde 72: 525-535). To confirm that Trichinella infection altered