Cytokine Profile Response of Human Peripheral Blood Mononuclear Cells Stimulated by Bartonella bacilliformis.

Carrion's disease is a neglected endemic disease found in remote Andean areas. As an overlooked disease, knowledge of innate immune responses to Bartonella bacilliformis, the etiological agent, is scarce. This study aimed to evaluate the cytokine response to B. bacilliformis using in vitro human peripheral blood mononuclear cells (PBMCs) stimulations. PBMCs from naive adults were isolated by gradient centrifugation and cocultured with heat-inactivated (HI) B. bacilliformis at different incubation times (3, 6, 12, 24, and 36 h). Cytokines, chemokines, and growth factors were determined in culture supernatants by multiplex fluorescent bead-based quantitative suspension array technology. During the first 36 h, a proinflammatory response was observed, including tumor necrosis factor-α, interleukin (IL)-1α, IL-1ß, interferon-α2, and IL-6, followed by an anti-inflammatory response mainly related to IL-1RA. Moreover, high expression levels of chemokines IL-8, monocyte chemoattractant protein-1α, and macrophage inflammatory protein (MIP)-1ß were detected from 3 h poststimulation and MIP-1α was detected at 24 h. Some growth factors, mainly granulocyte macrophage colony-stimulating factor and granulocyte colony-stimulating factor, and in minor concentrations vascular endothelial growth factor, epidermal growth factor, and eotaxin, were also detected. Innate response to HI B. bacilliformis stimulation consists of a rapid and strong proinflammatory response characterized by a wide range of cytokines and chemokines followed by an anti-inflammatory response and increased specific growth factors.

Dubious presence of Bartonella bacilliformis in ticks from Madre de Dios, Peru.

Bartonella bacilliformis has recently been described in Amblyomma scalpturatum, Amblyomma ovale and Rhipicephalus microplus collected from wild animals in the Peruvian region of Madre de Dios. In this communication, I will discuss the results of a recent study by del Valle-Mendoza et al. together with the B. bacilliformis epidemiology. Following my argumentation, I consider the presence of this microorganism in the above ticks improbable.

Carrion's disease: an eradicable illness?

Carrion's disease is a neglected tropical disease caused by Bartonella bacilliformis, a vector-borne pathogen restricted to the Andean valleys of Peru, Ecuador and Colombia. Carrion's disease is a biphasic illness; in the acute phase the case-fatality rate can be as high as 88 %, related to high parasitemia, arriving to almost all erythrocytes, and secondary bacterial infections close related with the development of transient immunosuppression in the earlier illness phases. In addition, there are an undefined number of asymptomatic carriers that are reservoirs of the etiological agent of Carrion's disease in endemic areas, they make take into account due to they are the perpetuators of this disease. The actual scenario of Carrion's disease, in which the illness is arriving to new areas, due to the expansion of the vector's distribution, suggests that now may be a crucial time to design a strategy focusing on its elimination.

Colonization of Lutzomyia verrucarum and Lutzomyia longipalpis Sand Flies (Diptera: Psychodidae) by Bartonella bacilliformis, the Etiologic Agent of Carrión's Disease.

Bartonella bacilliformis is a pathogenic bacterium transmitted to humans presumably by bites of phlebotomine sand flies, infection with which results in a bi-phasic syndrome termed Carrión's disease. After constructing a low-passage GFP-labeled strain of B. bacilliformis, we artificially infected Lutzomyia verrucarum and L. longipalpis populations, and subsequently monitored colonization of sand flies by fluorescence microscopy. Initially, colonization of the two fly species was indistinguishable, with bacteria exhibiting a high degree of motility, yet still confined to the abdominal midgut. After 48 h, B. bacilliformis transitioned from bacillus-shape to a non-motile, small coccoid form and appeared to be digested along with the blood meal in both fly species. Differences in colonization patterns became evident at 72 h when B. bacilliformis was observed at relatively high density outside the peritrophic membrane in the lumen of the midgut in L. verrucarum, but colonization of L. longipalpis was limited to the blood meal within the intra-peritrophic space of the abdominal midgut, and the majority of bacteria were digested along with the blood meal by day 7. The viability of B. bacilliformis in L. longipalpis was assessed by artificially infecting, homogenizing, and plating for determination of colony-forming units in individual flies over a 13-d time course. Bacteria remained viable at relatively high density for approximately seven days, suggesting that L. longipalpis could potentially serve as a vector. The capacity of L. longipalpis to transmit viable B. bacilliformis from infected to uninfected meals was analyzed via interrupted feeds. No viable bacteria were retrieved from uninfected blood meals in these experiments. This study provides significant information toward understanding colonization of sand flies by B. bacilliformis and also demonstrates the utility of L. longipalpis as a user-friendly, live-vector model system for studying this severely neglected tropical disease.

The population structure of Lutzomyia verrucarum (Diptera: Psycodidae), a Bartonella bacilliformis and Leishmania peruviana vector in Peru.

The population genetic structure of Lutzomyia verrucarum (Townsend), a sand fly disease vector of Carrion's disease and cutaneous leishmaniasis in the Peruvian Andes, was characterized by sequencing 653 bp of cytochrome b and 1,125 bp of the NADH dehydrogenase subunit 4 genes of its mitochondrial genome. DNA sequence variation within and between valleys was compared in a sample of 220 sand flies from three valleys (Purisima, Huaylas, and Conchucos) and five departments (Amazonas, Cajamarca, Piura, Lima, and Huancavelica). Gene network and phylogenetic analyses indicated a high similarity of haplotypes collected within a single valley (0-0.52% nucleotide divergence). Flies from each valley had unique genotypes not shared with specimens from other valleys or from more distant regions (0.8-3.1% nucleotide divergence). Mountain ranges and geographic distance appear to have impeded migration (N(m) = < 0.18) between valleys and separated populations into discrete genetic units.

Bartonella bacilliformis GroEL: effect on growth of human vascular endothelial cells in infected cocultures.

Bartonella are the only bacteria known to induce angioproliferative lesions of the human vasculature and liver during infection. Previous work from our lab suggests that GroEL participates in the mitogenic response observed in HUVEC cultures supplemented with the soluble fraction of Bartonella bacilliformis. Work in this study shows that exposure to high concentrations of the fraction is actually cytotoxic for HUVECs. To analyze this phenomenon, live B. bacilliformis-HUVEC cocultures were employed to study the effect of excess bacterial GroEL on the host cell during active infection. Four B. bacilliformis strains were generated to produce varying levels of GroEL. HUVEC cocultures with LSS100, a strain that synthesizes markedly greater quantities of GroEL relative to others, significantly accelerates apoptosis of the cocultured HUVECs relative to other strains. Acceleration of apoptosis can be inhibited by Z-VAD-FMK, a pan-caspase inhibitor. Time course data show that, at 18 h of infection, both LSS100 and control strains significantly inhibit spontaneous apoptosis of cocultured HUVECs, as previously reported for other Bartonella species. However, by 48 h, LSS100 significantly increases apoptosis of the host cell. We hypothesize that intracellular Bartonella GroEL functions as an Hsp60 analogue, a eukaryotic orthologue known to accelerate pro-caspase 3 activation by enhancing its vulnerability to upstream activator caspases. These data suggest another strategy whereby Bartonella may regulate host cell growth.