The existence of intracellular rickettsiae requires entry, survival, and replication in the eukaryotic host cells and exit to initiate new infection. While endothelial cells are the preferred target cells for most pathogenic rickettsiae, infection of monocytes/macrophages may also contribute to the establishment of rickettsial infection and resulting pathogenesis. We initiated studies to characterize macrophage-Rickettsia akari and -Rickettsia typhi interactions and to determine how rickettsiae survive within phagocytic cells. Flow cytometry, microscopic analysis, and LDH release demonstrated that R. akari and R. typhi caused negligible cytotoxicity in mouse peritoneal macrophages as well as in macrophage-like cell line, P388D1. Host cells responded to rickettsial infection with increased secretion of proinflammatory cytokines such as interleukin-1␤ (IL-1␤) and IL-6. Furthermore, macrophage infection with R. akari and R. typhi resulted in differential synthesis and expression of IL-␤ and IL-6, which may correlate with the existence of biological differences among these two closely related bacteria. In contrast, levels of gamma interferon (IFN-␥), IL-10, and IL-12 in supernatants of infected P388D1 cells and mouse peritoneal macrophages did not change significantly during the course of infection and remained below the enzyme-linked immunosorbent assay cytokine detection limits. In addition, differential expression of cytokines was observed between R. akari-and R. typhi-infected macrophages, which may correlate with the biological differences among these closely related bacteria.

The genomic sequence of Rickettsia prowazekii, the obligate intracellular bacterium responsible for epidemic typhus, reveals an uncharacterized invasion gene homolog (invA). The deduced protein of 18,752 Da contains a Nudix signature, the specific motif found in the Nudix hydrolase family. To characterize the function of InvA, the gene was cloned and overexpressed in Escherichia coli. The expressed protein was purified to near homogeneity and subsequently tested for its enzymatic activity against a series of nucleoside diphosphate derivatives. The purified InvA exhibits hydrolytic activity toward dinucleoside oligophosphates (Np n N; n > 5), a group of cellular signaling molecules. At optimal pH 8.5, the enzyme actively degrades adenosine (

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Multiple genetic subtypes of human immunodeficiency virus type 1 (HIV-1) have been identified among internationally collected isolates. The HIV-1 epidemic in Thailand is largely due to B and E subtypes of virus. Dual infection with distinct HIV-1 subtypes would suggest that antiviral immunity evoked by one subtype can be incompletely protective against a second. Polymerase chain reaction typing and serologic typing were used to screen a panel of specimens from HIV-1-infected subjects in Thailand. Two persons simultaneously harbored HIV-1 of env subtypes B and E, and this was confirmed by colony hybridization with subtype-specific probes and nucleotide sequence analysis of a 630-bp fragment of gp120 from multiple molecular clones. In addition, both subtypes were identified in cocultured peripheral blood mononuclear cells from 1 individual. These data provide the first evidence of dual HIV-1 infection in humans and reinforce the need for polyvalent vaccines.

The first tissue culture isolates of the unique Anaplasma phagocytophilum strain, Ap-Variant 1, were obtained in the Ixodes scapularis tick-derived cell line ISE6. Two isolates were from goat blood samples: one from a goat infected with I. scapularis ticks from Rhode Island and a second from a goat infected by serial passage of blood from the first infected goat. Eight isolates were made directly from I. scapularis ticks collected from white-tailed deer in Minnesota and represent the first isolations of an Anaplasma species directly from ticks. Each of the 10 isolates had a 16S rRNA gene sequence identical to that previously described for Ap-Variant 1, but differences within the ank gene were found that suggest natural variation. Prevalence of Anaplasma in the Minnesota ticks was 63.9%; 23 of 36 ticks tested by PCR were positive. Six of the tick-derived isolates were obtained from a set of 18 PCR-positive ticks, for a 33.3% isolation success rate. The conservation of host tropism among the Rhode Island and Minnesota isolates of Ap-Variant 1 was examined by use of experimental infections of mice and a goat. A Minnesota tick-derived isolate (MN-61-2) was used to inoculate naïve animals, and this isolate was able to infect a goat but unable to infect each of five mice, confirming that the Minnesota isolates have the same host tropism as Ap-Variant 1 from the northeastern United States. Light and electron microscopy of the Ap-Variant 1 isolate MN-61-2 in ISE6 cells showed cytoplasmic inclusions characteristic of A. phagocytophilum with pleomorphic bacteria in membrane-bound vacuoles and both electron-dense and electron-lucent forms.

During a scrub typhus outbreak investigation in Thailand, 4 isolates of O. tsutsugamushi were obtained and established in culture. Phylogenetic analysis based on the 56-kDa type-specific antigen gene demonstrated that the isolates fell into 4 genetic clusters, 3 of which had been previously reported and 1 that represents a new genotype.

Scrub typhus, caused by antigenically disparate isolates of Orientia tsutsugamushi, is a widely distributed mite-borne human disease in the Asia Pacific region. Information regarding the heterogeneity of the immunodominant 56-kDa type-specific antigen (TSA) gene is crucial for the design and evaluation of scrub typhusspecific diagnostic assays and vaccines. Using indirect immunofluorescence assays (IFA) and PCR assays, O. tsutsugamushi was detected samples from rodents and patients with fever of unknown origin obtained from six provinces of Thailand during 2004 to 2007. Sequences were determined for a fragment of the 56-kDa TSA gene, and the relationship between these sequences and those previously determined were assessed. The phylogenetic analyses of partial 56-kDa TSA gene sequences demonstrated wide diversity and distribution of O. tsutsugamushi genotypes in Thailand. Furthermore, the genetic diversity grouped the scrub typhus agents into two commonly and five infrequently found genotypes within six provinces of Thailand. The two most commonly found genotypes of O. tsutsugamushi described in this study do not associate with the prototype strains that are widely used for the design and evaluation of diagnostic assays and vaccine candidates. Thus, these new genotypes should be considered for future scrub typhus assay and vaccine development.

Flea and tick specimens (5-10 fleas or ticks) on dogs and cats from various sites in Bangkok were tested by polymerase chain reaction and DNA sequencing to detect DNA of bacteria Rickettsia (gltA and 17 kDa genes), Anaplasmataceae (16S rRNA gene), and Bartonella (pap31 and its genes). We confirmed that Rickettsia sp. related to Rickettsia felis was detected in 66 of 98 (67.4%) flea specimens from dogs, whereas 8 Bartonella henselae and 2 Bartonella clarridgeiae were detected in 10 of 54 (18.5%) flea specimens from cats. Further, this work provides the first evidence of 10 Ehrlichia canis (3.3%), 7 Anaplasma platys (2.3%), and 2 Wolbachia spp. (0.66%) in 304 Rhipicephalus sanguineus tick specimens in Thailand.