Diversity of Anaplasma phagocytophilum Strains, USA

We analyzed the structure of the expression site encoding the immunoprotective protein MSP2/P44 from multiple Anaplasma phagocytophilum strains in the United States. The sequence of p44ESup1 had diverged in Ap-variant 1 strains infecting ruminants. In contrast, no differences were detected between A. phagocytophilum strains infecting humans and domestic dogs

IS1111 insertion sequences of Coxiella burnetii: characterization and use for repetitive element PCR-based differentiation of Coxiella burnetii isolates

<p>Abstract</p> <p>Background</p> <p><it>Coxiella burnetii </it>contains the IS<it>1111 </it>transposase which is present 20 times in the Nine Mile phase I (9Mi/I) genome. A single PCR primer that binds to each IS element, and primers specific to a region ~500-bp upstream of each of the 20 IS<it>1111 </it>elements were designed. The amplified products were characterized and used to develop a repetitive element PCR genotyping method.</p> <p>Results</p> <p>Isolates Nine Mile phase II, Nine Mile RSA 514, Nine Mile Baca, Scottish, Ohio, Australian QD, Henzerling phase I, Henzerling phase II, M44, KAV, PAV, Q238, Q195 and WAV were tested by PCR and compared to 9Mi/I. Sequencing was used to determine the exact differences in isolates which lacked specific IS elements or produced PCR products of differing size. From this data, an algorithm was created utilizing four primer pairs that allows for differentiation of unknown isolates into five genomic groups. Additional isolates (Priscilla Q177, Idaho Q, Qiyi, Poker Cat, Q229 and Q172) and nine veterinary samples were characterized using the algorithm which resulted in their placement into three distinct genomic groups.</p> <p>Conclusion</p> <p>Through this study significant differences, including missing elements and sequence alterations within and near IS element coding regions, were found between the isolates tested. Further, a method for differentiation of <it>C. burnetii </it>isolates into one of five genomic groups was created. This algorithm may ultimately help to determine the relatedness between known and unknown isolates of <it>C. burnetii</it>.</p

Anaplasma phagocytophilum in White-tailed Deer

We examined the reservoir potential of white-tailed deer for Anaplasma phagocytophilum. Results suggest that white-tailed deer harbor a variant strain not associated with human infection, but contrary to published reports, white-tailed deer are not a reservoir for strains that cause human disease. These results will affect surveillance studies of vector and reservoir populations

On Rickettsia Nomenclature

On Rickettsia Nomenclatur

Serologic and Molecular Detection of Granulocytic Ethrlichiosis in Rhode Island

A new indirect fluorescent-antibody (IFA) assay with antigen produced in vitro in the human promyelocytic leukemia cell line HL60 was used to identify the first recognized case of human granulocytic ehrlichiosis in Rhode Island. This IFA assay was used to detect granulocytic ehrlichiae in white-footed mice and in a dog inhabiting the area surrounding the patient’s residence. Host-seeking Ixodes scapularis ticks found in the same habitat also were infected. I. scapularis ticks collected from other locations were fed on dogs and New Zealand White rabbits to assess the competency of these species as hosts of granulocytotropic Ehrlichia. Tick-induced infections of dogs were confirmed by serologic testing, tissue culture isolation, and PCR amplification, whereas several rabbits seroconverted but were PCR and culture negative. PCR amplification of the 16S rRNA gene and DNA sequencing of the PCR products or culture isolation was used to confirm granulocytic Ehrlichia infections in humans, dogs, white-footed mice, and ticks

Outcome of Diagnostic Tests Using Samples from Patients with Culture-Proven Human Monocytic Ehrlichiosis: Implications for Surveillance

We describe the concordance among results from various laboratory tests using samples derived from nine culture-proven cases of human monocytic ehrlichiosis (HME) caused by Ehrlichia chaffeensis. A class-specific indirect immunofluorescence assay for immunoglobulin M (IgM) and IgG, using E. chaffeensis antigen, identified 44 and 33% of the isolation-confirmed HME patients on the basis of samples obtained at initial clinical presentation, respectively; detection of morulae in blood smears was similarly insensitive (22% positive). PCR amplifications of ehrlichial DNA targeting the 16S rRNA gene, the variable-length PCR target gene, and the groESL operon were positive for whole blood specimens obtained from all patients at initial presentation. As most case definitions of HME require a serologic response with compatible illness for a categorization of even probable disease, PCR would have been required to confirm the diagnosis of HME in all nine of these patients without the submission of a convalescent-phase serum sample. These data suggest that many, if not most, cases of HME in patients who present early in the course of the disease may be missed and underscore the limitations of serologically based surveillance systems

Anaplasma phagocytophilum and Anaplasma marginale Elicit Different Gene Expression Responses in Cultured Tick Cells

The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes obligate tick-transmitted intracellular organisms, Anaplasma phagocytophilum and Anaplasma marginale that multiply in both vertebrate and tick host cells. Recently, we showed that A. marginale affects the expression of tick genes that are involved in tick survival and pathogen infection and multiplication. However, the gene expression profile in A. phagocytophilum-infected tick cells is currently poorly characterized. The objectives of this study were to characterize tick gene expression profile in Ixodes scapularis ticks and cultured ISE6 cells in response to infection with A. phagocypthilum and to compare tick gene expression responses in A. phagocytophilum- and A. marginale-infected tick cells by microarray and real-time RT-PCR analyses. The results of these studies demonstrated modulation of tick gene expression by A. phagocytophilum and provided evidence of different gene expression responses in tick cells infected with A. phagocytophilum and A. marginale. These differences in Anaplasma-tick interactions may reflect differences in pathogen life cycle in the tick cells

Genetic Variants of Ehrlichia phagocytophila1, Rhode Island and Connecticut

Primers were used to amplify a 561-bp region of the 16S rRNA gene of Ehrlichia phagocytophila from Ixodes scapularis ticks and small mammals collected in Rhode Island and Connecticut. DNA sequences for all 50 E. phagocytophila-positive samples collected from 1996 through 1998 in southwestern Connecticut were identical to the sequence reported for E. phagocytophila DNA from confirmed human cases. In contrast, the sequences from 92 of 123 E. phagocytophila-positive Rhode Island samples collected from 1996 through 1999 included several variants differing by 1-2 nucleotides from that in the agent infecting humans. While 11.9% of 67 E. phagocytophila-positive ticks collected during 1997 in Rhode Island harbored ehrlichiae with sequences identical to that of the human agent, 79.1% had a variant sequence not previously described. The low incidence of human ehrlichiosis in Rhode Island may in part result from interference by these variant ehrlichiae with maintenance and transmission of the true agent of human disease

Rapid typing of Coxiella burnetii

Coxiella burnetii has the potential to cause serious disease and is highly prevalent in the environment. Despite this, epidemiological data are sparse and isolate collections are typically small, rare, and difficult to share among laboratories as this pathogen is governed by select agent rules and fastidious to culture. With the advent of whole genome sequencing, some of this knowledge gap has been overcome by the development of genotyping schemes, however many of these methods are cumbersome and not readily transferable between institutions. As comparisons of the few existing collections can dramatically increase our knowledge of the evolution and phylogeography of the species, we aimed to facilitate such comparisons by extracting SNP signatures from past genotyping efforts and then incorporated these signatures into assays that quickly and easily define genotypes and phylogenetic groups. We found 91 polymorphisms (SNPs and indels) among multispacer sequence typing (MST) loci and designed 14 SNP-based assays that could be used to type samples based on previously established phylogenetic groups. These assays are rapid, inexpensive, real-time PCR assays whose results are unambiguous. Data from these assays allowed us to assign 43 previously untyped isolates to established genotypes and genomic groups. Furthermore, genotyping results based on assays from the signatures provided here are easily transferred between institutions, readily interpreted phylogenetically and simple to adapt to new genotyping technologies