Biofilm formation is the primary virulence factor of Staphylococcus epidermidis. S. epidermidis biofilms preferentially form on abiotic surfaces and may contain multiple matrix components, including proteins such as accumulation-associated protein (Aap). Following proteolytic cleavage of the A domain, which has been shown to enhance binding to host cells, B domain homotypic interactions support cell accumulation and biofilm formation. To further define the contribution of Aap to biofilm formation and infection, we constructed an aap allelic replacement mutant and an icaADBC aap double mutant. When subjected to fluid shear, strains deficient in Aap production produced significantly less biofilm than Aap-positive strains. To examine the in vivo relevance of our findings, we modified our previously described rat jugular catheter model and validated the importance of immunosuppression and the presence of a foreign body to the establishment of infection. The use of our allelic replacement mutants in the model revealed a significant decrease in bacterial recovery from the catheter and the blood in the absence of Aap, regardless of the production of polysaccharide intercellular adhesin (PIA), a well-characterized, robust matrix molecule. Complementation of the aap mutant with full-length Aap (containing the A domain), but not the B domain alone, increased initial attachment to microtiter plates, as did in trans expression of the A domain in adhesion-deficient Staphylococcus carnosus. These results demonstrate Aap contributes to S. epidermidis infection, which may in part be due to A domain-mediated attachment to abiotic surfaces.

We report here on a quantitative real-time reverse transcription-PCR (qRT-PCR) assay for assessing drug efficacy against the intracellular pathogen Cryptosporidium parvum. The qRT-PCR assay detects 18S rRNA transcripts from both parasites, that is, the cycle threshold for 18S rRNA from parasites (C T Cryptosporidium parvum is an intracellular parasitic protist that infects both humans and animals (5,22). This parasite belongs to the phylum Apicomplexa. However, recent phylogenetic reconstructions have suggested that the genus Cryptosporidium probably represents an early branch at the base of the phylum (31). Together with C. hominis (a new species recently renamed from type 1 C. parvum) (15), C. parvum is also a significant opportunistic pathogen among immunocompromised individuals (e.g., AIDS patients) (5). Currently, only a single drug (i.e., nitazoxanide [NTZ]) has been approved for the treatment of cryptosporidiosis in the United States and Central and South America (10; see also http://www.romark .com). Therefore, there is a need for the development of new anti-Cryptosporidium drugs.[In vitro drug testing is a critical step in the early stage of drug development. To evaluate drug effects against C. parvum in vitro, parasites must be cultured by infecting human or animal cells. Currently, several techniques are available for the evaluation of drug efficacy against C. parvum in vitro. These techniques include the microscopic method (by counting the number of fluorescently labeled or chemically stained parasites per microscopic field) (8), microtiter enzyme-linked immunosorbent assay (25, 26), chemiluminescence immunoassay (28, 29), and potentially real-time quantitative PCR (qPCR) (6, 13). While all methods may reliably detect the level of parasites in vitro, many have their own limitations. The microscopic method can be labor-intensive and highly subjective. Antibodybased techniques rely on the availability of good antibodies and are limited by the sensitive and limited signal-to-noise ratio (25). More recently, a real-time qPCR-based method has been introduced for assessment of the effects of drugs against C. parvum in vitro by detecting the level of parasite DNA, and this method promised a wide dynamic range of detection (6, 13). However, our recent observations suggest that the DNA level may not always be well correlated with the number of infected parasites because the relationship between the inoculated number of parasites and the normalized cycle thresholds (C T s) of detection by qPCR is not linear. Furthermore, the DNA from dead parasites may also exist in these samples (see below for details). In addition, the reported qPCR method displayed significant plate-to-plate variations (13), indicating that improvements are needed to take full advantage of the real-time PCR system for drug testing.Here we report on an improved method for assessment of drug efficacy against C. parvum infection in vitro, based on the quantitative real-time reverse transcription-PCR (qRT-PCR) technique. By detecting the level ...

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SummaryWe have discovered and analysed two novel, linear extrachromosomal double-stranded RNAs (dsRNAs) within oocysts of major north Amercian isolates of Cryptosporidium parvum, a parasitic protozoan that infects the gastrointestinal tract of a variety of mammals, including humans. These dsRNAs were found to reside within the cytoplasm of sporozoites, and were not detected in other species of the genus. cDNAs representing both dsRNA genomes were cloned and sequenced, 1786 and 1374 nt, and each encoded one large open reading frame (ORF). The deduced protein sequence of the larger dsRNA (L-dsRNA) had homology with viral RNA-dependent RNA polymerases (RDRP), with more similarity to polymerases from fungi than those from other protozoa. The deduced protein sequence from the smaller dsRNA (S-dsRNA) had limited similarity with mitogen-activated c-June NH 2 terminal protein kinases (JNK) from mammalian cells. Attempts to visually identify or purify virus-like particles associated with the dsRNAs were unsuccessful. Sensitivity of the dsRNAs to RNase A also suggests that the dsRNAs may be unencapsidated. A RDRP activity was identified in crude extracts from C. parvum sporozoites and products of RNA polymerase activity derived in vitro were similar to the dsRNAs purified directly from the parasites.

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