The role of CD8 T lymphocytes in rickettsial infections.

Arthropod-borne obligately intracellular bacteria pose a difficult challenge to the immune system. The genera Rickettsia, Orientia, Ehrlichia, and Anaplasma evolved mechanisms of immune evasion, and each interacts differently with the immune system. The roles of CD8 T cells include protective immunity and immunopathology. In Rickettsia infections, CD8 T cells are protective mediated in part by cytotoxicity toward infected cells. In contrast, TNF-α overproduction by CD8 T cells is pathogenic in lethal ehrlichiosis by induction of apoptosis/necrosis in hepatocytes. Yet, CD8 T cells, along with CD4 T cells and antibodies, also contribute to protective immunity in ehrlichial infections. In granulocytic anaplasmosis, CD8 T cells impact pathogen control modestly but could contribute to immunopathology by virtue of their dysfunction. While preliminary evidence indicates that CD8 T cells are important in protection against Orientia tsutsugamushi, mechanistic studies have been neglected. Valid animal models will enable experiments to elucidate protective and pathologic immune mechanisms. The public health need for vaccines against these agents of human disease, most clearly O. tsutsugamushi, and the veterinary diseases, canine monocytotropic ehrlichiosis (Ehrlichia canis), heartwater (Ehrlichia ruminantium), and bovine anaplasmosis (A. marginale), requires detailed immunity and immunopathology investigations, including the roles of CD8 T lymphocytes.

High virulence of Wolbachia after host switching: when autophagy hurts.

Wolbachia are widespread endosymbionts found in a large variety of arthropods. While these bacteria are generally transmitted vertically and exhibit weak virulence in their native hosts, a growing number of studies suggests that horizontal transfers of Wolbachia to new host species also occur frequently in nature. In transfer situations, virulence variations can be predicted since hosts and symbionts are not adapted to each other. Here, we describe a situation where a Wolbachia strain (wVulC) becomes a pathogen when transfected from its native terrestrial isopod host species (Armadillidium vulgare) to another species (Porcellio d. dilatatus). Such transfer of wVulC kills all recipient animals within 75 days. Before death, animals suffer symptoms such as growth slowdown and nervous system disorders. Neither those symptoms nor mortalities were observed after injection of wVulC into its native host A. vulgare. Analyses of wVulC's densities in main organs including Central Nervous System (CNS) of both naturally infected A. vulgare and transfected P. d. dilatatus and A. vulgare individuals revealed a similar pattern of host colonization suggesting an overall similar resistance of both host species towards this bacterium. However, for only P. d. dilatatus, we observed drastic accumulations of autophagic vesicles and vacuoles in the nerve cells and adipocytes of the CNS from individuals infected by wVulC. The symptoms and mortalities could therefore be explained by this huge autophagic response against wVulC in P. d. dilatatus cells that is not triggered in A. vulgare. Our results show that Wolbachia (wVulC) can lead to a pathogenic interaction when transferred horizontally into species that are phylogenetically close to their native hosts. This change in virulence likely results from the autophagic response of the host, strongly altering its tolerance to the symbiont and turning it into a deadly pathogen.

New insight into immunity and immunopathology of Rickettsial diseases.

Human rickettsial diseases comprise a variety of clinical entities caused by microorganisms belonging to the genera Rickettsia, Orientia, Ehrlichia, and Anaplasma. These microorganisms are characterized by a strictly intracellular location which has, for long, impaired their detailed study. In this paper, the critical steps taken by these microorganisms to play their pathogenic roles are discussed in detail on the basis of recent advances in our understanding of molecular Rickettsia-host interactions, preferential target cells, virulence mechanisms, three-dimensional structures of bacteria effector proteins, upstream signalling pathways and signal transduction systems, and modulation of gene expression. The roles of innate and adaptive immune responses are discussed, and potential new targets for therapies to block host-pathogen interactions and pathogen virulence mechanisms are considered.

Wolbachia-induced cytoplasmic incompatibility is associated with decreased Hira expression in male Drosophila.

BACKGROUND: Wolbachia are obligate endosymbiotic bacteria that infect numerous species of arthropods and nematodes. Wolbachia can induce several reproductive phenotypes in their insect hosts including feminization, male-killing, parthenogenesis and cytoplasmic incompatibility (CI). CI is the most common phenotype and occurs when Wolbachia-infected males mate with uninfected females resulting in no or very low numbers of viable offspring. However, matings between males and females infected with the same strain of Wolbachia result in viable progeny. Despite substantial scientific effort, the molecular mechanisms underlying CI are currently unknown. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression studies were undertaken in Drosophila melanogaster and D. simulans which display differential levels of CI using quantitative RT-PCR. We show that Hira expression is correlated with the induction of CI and occurs in a sex-specific manner. Hira expression is significantly lower in males which induce strong CI when compared to males inducing no CI or Wolbachia-uninfected males. A reduction in Hira expression is also observed in 1-day-old males that induce stronger CI compared to 5-day-old males that induce weak or no CI. In addition, Hira mutated D. melanogaster males mated to uninfected females result in significantly decreased hatch rates comparing with uninfected crosses. Interestingly, wMel-infected females may rescue the hatch rates. An obvious CI phenotype with chromatin bridges are observed in the early embryo resulting from Hira mutant fertilization, which strongly mimics the defects associated with CI. CONCLUSIONS/SIGNIFICANCE: Our results suggest Wolbachia-induced CI in Drosophila occurs due to a reduction in Hira expression in Wolbachia-infected males leading to detrimental effects on sperm fertility resulting in embryo lethality. These results may help determine the underlying mechanism of CI and provide further insight in to the important role Hira plays in the interaction of Wolbachia and its insect host.

Wolbachia interferes with ferritin expression and iron metabolism in insects.

Wolbachia is an intracellular bacterium generally described as being a facultative reproductive parasite. However, Wolbachia is necessary for oogenesis completion in the wasp Asobara tabida. This dependence has evolved recently as a result of interference with apoptosis during oogenesis. Through comparative transcriptomics between symbiotic and aposymbiotic individuals, we observed a differential expression of ferritin, which forms a complex involved in iron storage. Iron is an essential element that is in limited supply in the cell. However, it is also a highly toxic precursor of Reactive Oxygen Species (ROS). Ferritin has also been shown to play a key role in host-pathogen interactions. Measuring ferritin by quantitative RT-PCR, we confirmed that ferritin was upregulated in aposymbiotic compared to symbiotic individuals. Manipulating the iron content in the diet, we showed that iron overload markedly affected wasp development and induced apoptotic processes during oogenesis in A. tabida, suggesting that the regulation of iron homeostasis may also be related to the obligate dependence of the wasp. Finally, we demonstrated that iron metabolism is influenced by the presence of Wolbachia not only in the obligate mutualism with A. tabida, but also in facultative parasitism involving Drosophila simulans and in Aedes aegypti cells. In these latter cases, the expression of Wolbachia bacterioferritin was also increased in the presence of iron, showing that Wolbachia responds to the concentration of iron. Our results indicate that Wolbachia may generally interfere with iron metabolism. The high affinity of Wolbachia for iron might be due to physiological requirement of the bacterium, but it could also be what allows the symbiont to persist in the organism by reducing the labile iron concentration, thus protecting the cell from oxidative stress and apoptosis. These findings also reinforce the idea that pathogenic, parasitic and mutualistic intracellular bacteria all use the same molecular mechanisms to survive and replicate within host cells. By impacting the general physiology of the host, the presence of a symbiont may select for host compensatory mechanisms, which extends the possible consequences of persistent endosymbiont on the evolution of their hosts.

Monoclonal antibody-based immunohistochemical diagnosis of rickettsialpox: the macrophage is the principal target.

Cutaneous biopsies of five eschars and two rash lesions from five patients from New York City with documented rickettsialpox were examined by immunohistochemical methods with a monoclonal antibody directed against spotted fever group rickettsial lipopolysaccharide for the presence and cellular location of Rickettsia akari Rickettsiae were identified in all of the five patients, with good concordance of results for the same biopsy tissues with previously reported results by the direct immunofluorescence method. In contrast with immunofluorescence, which did not reveal the location of the organisms, immunohistochemical examination demonstrated R. akari to be in perivascular cells, morphologically resembling macrophages. Evaluation with double staining for rickettsiae and either CD68 or Factor VIII-related antigen revealed that the predominant infected cell type was CD68-positive macrophages, and only a rare rickettsia was detected in vascular endothelium, the major target cell for other rickettsioses. These results provide a diagnostic method for rickettsialpox and other spotted fever group rickettsioses and indicate that the elucidation of the pathogenesis of rickettsialpox must take into account that its target cell differs from that of Rocky Mountain spotted fever, boutonneuse fever, louse-borne typhus fever, and murine typhus.

Effect of tick-borne fever (Ehrlichia phagocytophila) and trypanosomiasis (Trypanosoma brucei 1066) on the pharmacokinetics of sulfadimidine and its metabolites in goats.

The effect of tick-borne fever (TBF) and trypanosomiasis (TBR) on the plasma disposition of sulfadimidine (SDD) in goats was studied after iv administration of 20 and 200 mg/kg of body weight. In each group of six goats, the plasma disappearance curves showed four animals with rapid and two with slow SDD elimination. It is likely that this difference is determined by oxidative rather than acetylation phenotype. In all goats administered 20 mg/kg, half-life increased with TBF but not with TBR. Vd(beta) decreased with both infections. With 200 mg/kg, Vd did not change, whereas AUC and MRT increased with both infections. Metabolites were examined in TBF experiments: N4-acetyl-SDD (N4Ac), 6-hydroxymethyl-SDD (CH2OH) and its glucuronide, 5-hydroxy-SDD (SOH) and its glucuronide, and 6-carboxy-SDD (COOH) and its glucuronide (COOH-glu). At low dose (20 mg/kg), TBF caused the proportion of dose recovered from urine as unchanged SDD to be halved, whereas N4Ac increased correspondingly (2x). After the high dose (200 mg/kg), elimination was saturated and changes in proportional recovery of SDD in urine were less. However, the N4Ac proportion was still doubled, in contrast to the other metabolites, suggesting that saturation was caused by oxidations rather than by acetylation. Formation of CH2OH was the same in health and disease after the low dose, but glucuronidation dropped from 20% to 4% (rapid) or 7% (slow) of total CH2OH.(ABSTRACT TRUNCATED AT 250 WORDS)

Some pharmacokinetic data of aditoprim and trimethoprim in healthy and tick-borne fever infected dwarf goats.

Aditoprim (AP) is a new dihydrofolate reductase inhibitor, which is structurally related to trimethoprim (TMP). The pharmacokinetics of AP (10 mg/kg) and TMP (20 mg/kg) were assessed in healthy dwarf goats. Therapeutic efficacy against rickettsial infections was tested in tick-borne fever (TBF) infected goats. The animals were given TMP (n = 5) or AP (n = 5) by i.v. injection, and subsequently the drugs were administered orally (same groups, similar doses). Finally, both groups were infected with TBF and the i.v. experiment was repeated. Plasma concentration-time curves for both drugs followed first-order two-compartment decay. For TMP, mean t1/2 beta +/- SEM (h) was 0.84 +/- 0.06 (i.v. control) and 0.90 +/- 0.06 (i.v. infected), respectively, whereas for AP values of 8.00 +/- 0.31 (i.v. control) and 10.28 +/- 0.67 (i.v. infected) were obtained (P less than 0.05). Mean Vd beta +/- SEM values (l/kg) were 3.84 +/- 0.27 (i.v. control) and 4.07 +/- 0.85 (i.v. infected) for TMP (NS) and 7.02 +/- 0.63 vs 9.29 +/- 0.21 (P less than 0.05) for AP. After i.v. injection, rumen fluid concentrations of AP were significantly (P less than 0.05) higher and more persistent than those of TMP. For AP, the plasma and rumen fluid concentrations at 3 h were 1.20 +/- 0.06 micrograms/ml and 0.85 +/- 0.17 microgram/ml, respectively. After oral administration of TMP, Cmax in plasma was 0.12 +/- 0.01 microgram/ml and the maximum was reached after 1.2 +/- 0.16 h; systemic bioavailability (F) was 10.3% (relative to AUC i.v.). Oral treatment with AP resulted in a Cmax value of 0.21 +/- 0.02 microgram/ml with Tmax of 22.5 +/- 1.65 h and a F value of 71%. Based on WBC, serum ALP and rectal temperature responses, it was concluded that both TMP and AP were inactive against Ehrlichia phagocytophila.

The effect of tick-borne fever on metabolism and renal clearance of sulfadimidine in goats.

The tick-borne fever (TBF) model was used to study the effect of fever on the metabolism of sulfadimidine in goats. During TBF the elimination half-lives were prolonged, and the renal clearance values of sulfadimidine and the majority of its metabolites were markedly diminished compared with those in the uninfected state. During TBF the steady-state levels of the hydroxy metabolites were markedly increased. TBF reduced the extent of hydroxymethylation of the pyrimidine side chain; TBF did not affect acetylation of sulfadimidine. In one goat a progressive accumulation of the metabolites was noticed.

Chemotherapy and pharmacokinetics of some antimicrobial agents in healthy dwarf goats and those infected with Ehrlichia phagocytophila (tick-borne fever).

The therapeutic efficacy and pharmacokinetics of oxytetracycline (10 mg kg-1), ampicillin (20 mg kg-1) and a combination (TSS) of trimethoprim (20 mg kg-1), sulphadimidine (50 mg kg-1) and sulphamethylphenazole (50 mg kg-1) were investigated in normal dwarf goats and in those infected with Ehrlichia phagocytophila. Goats given oxytetracycline or TSS intravenously showed improvement, whereas ampicillin was ineffective. The infected goats had significantly prolonged elimination half-life values for sulphadimidine and oxytetracycline. The disposition kinetics of ampicillin and sulphamethylphenazole showed no marked differences between the healthy and infected animals. The tick-borne fever model used in the present study can be of value in testing the therapeutic efficacy and pharmacokinetics of chemotherapeutic agents in rickettsial infections.

Effect of tick-borne fever on the disposition of sulphadimidine and its metabolites in plasma of goats.

The effect of tick-borne fever (TBF) on the plasma disposition of sulphadimidine (SDM) and its metabolites in goats was studied. In uninfected goats, SDM was extensively metabolised mainly by hydroxylation, glucuronidation and to a minor extent by acetylation. In TBF infected goats the hydroxylation of SDM into 6-methylhydroxysulphadimidine (SCH2OH) as well as into 5-hydroxysulphadimidine (SOH) was markedly reduced (-57.6 and -63.6 per cent, respectively). An unidentified metabolite (metabolite X) was detected, which was largely glucuronidated in the uninfected goats. In the TBF infected goats the glucuronide derivatives of the X metabolite and of SOH were barely detectable. In TBF infected goats the plasma concentration of the N4-acetylated metabolite (N4-SDM) was decreased to a lesser extent (-22.1 per cent) than the hydroxy metabolites. Due to the diminished metabolism the elimination half-life of SDM was increased 1.8 times and the total sulphonamide body clearance was diminished compared with findings in the control experiments.