Babesia spp. are obligate protozoan parasites of red blood cells. Transmission to humans occurs through bites from infected ticks or blood transfusion. Infections with B. microti account for the majority of the reported cases of human babesiosis in the USA. A lower incidence is caused by the more recently described species B. duncani. The current gold standard for detection of Babesia is microscopic examination of blood smears. Recent PCR-based assays, including real-time PCR, have been developed for B. microti. On the other hand, molecular assays that detect and distinguish between B. microti and B. duncani infections are lacking. Closely related species of Babesia can be differentiated due to sequence variation within the internal transcribed spacer (ITS) regions of nuclear ribosomal RNAs. In the present study, we targeted the ITS regions of B. microti and B. duncani to develop sensitive and species-specific droplet digital PCR (ddPCR) assays. The assays were shown to discriminate B. microti from B. duncani and resulted in limits of detection of ~10 gene copies. Moreover, ddPCR for these species were useful in DNA extracted from blood of experimentally infected hamsters, detecting infections of low parasitemia that were negative by microscopic examination. In summary, we have developed sensitive and specific quantitative ddPCR assays for the detection of B. microti and B. duncani in blood. Our methods could be used as sensitive approaches to monitor the progression of parasitemia in rodent models of infection as well as serve as suitable molecular tests in blood screening.
The passive infusion of antibodies elicited in rabbits with a detoxified J5 lipopolysaccharide (LPS)/group B meningococcal outer membrane protein complex vaccine protected neutropenic rats from heterologous lethal gram-negative bacterial infection. In this study, active immunization was studied in neutropenic rats infected with Pseudomonas aeruginosa, in the presence or absence of ceftazidime therapy, and with Klebsiella pneumoniae. This vaccine elicited a > 200-fold increase in anti-J5 LPS antibody, which remained elevated throughout the duration of cyclophosphamide-induced neutropenia and for < or = 3 months. There was improved survival among immunized versus control animals: 48% (13/28) versus 7% (2/29) in Pseudomonas-challenged rats; 61% (11/18) versus 0% (0/10) in Pseudomonas- and ceftazidime-treated rats; and 64% (9/14) versus 13% (2/15) in Klebsiella-challenged rats (P < 0.01 for each comparison). Immunized animals had lower levels of bacteria in organs and lower levels of circulating endotoxin at the onset of fever. In conclusion, active immunization with an anti-endotoxin vaccine improved survival after infection with > or = 2 heterologous, clinically relevant bacterial species in immunocompromised animals. Active immunization with this vaccine merits further investigation.
The majority of strains of Toxoplasma gondii belong to three distinct clonal lines known as types I, II, and III. The outcome of the immune response to infection is influenced by the parasite strain type. The goal of this study was to examine differences in the kinetics of gene expression in microglial cells infected with types I, II, or III of T. gondii. In addition, a requirement for the integrity of host Toll-like receptor (TLR) signaling in parasite-mediated changes in gene expression was evaluated. Wild type murine microglial cells infected with T. gondii displayed different kinetic patterns of pro-inflammatory cytokine expression that were dependent on the parasite strain type. In general, types II and III elicited higher sustained responses compared to type I which induced fluctuating patterns of cytokine gene expression. Contrary to this, differences in the induction of anti-apoptotic gene expression were minimal among the different type strains throughout infection. Experiments with cells lacking the TLR adaptor molecules MAL and Myd88 showed a dependency on these factors for the pro-inflammatory response but not the anti-apoptotic response. The results show that the outcome of gene expression in T. gondii-infected microglial cells is dependent on the parasite strain type in a time-dependent manner and is selective to particular subsets of genes. The induction of an anti-apoptotic response by T. gondii infection in the absence of TLR signaling reflects a complex level of modulation of host functions by the parasite.
We describe the isolation of a mycobacterium from Acanthamoeba castellanii strain Ma (ATCC(®) 50370(™)). The mycobacterium resides within vacuoles of A. castellanii, can be cultured by routine methodologies, and is a member of the Mycobacterium avium complex. Previously unrecognized mycobacterial endosymbionts are likely common among strains of Acanthamoeba housed at culture collections.
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