Microarray technologies for gene transcript analysis in pancreatic cancer.

Pancreatic cancer is a devastating condition that is most often characterized by a poor prognosis. Microarray technologies are promising screening methods for the identification of potential markers for early diagnosis and chemotherapeutic intervention. In this article, we review the current state of pancreatic cancer research as it relates to the measurement of gene transcript levels by DNA microarray analysis.

mRNA expression in rabbit experimental aneurysms: a study using gene chip microarrays.

BACKGROUND AND PURPOSE: The molecular characteristics of intracranial aneurysms are still poorly documented. A rabbit elastase aneurysm model has been helpful in the evaluation of devices and strategies involved in endovascular treatment of aneurysms. The goal of this project was to document the molecular changes, assessed by gene chip microarrays, associated with the creation of aneurysms in this model compared with the contralateral carotid artery. MATERIALS AND METHODS: A microarray of rabbit genes of interest was constructed using rabbit nucleotide sequences from GenBank. Elastase-induced saccular aneurysms were created at the origin of the right common carotid artery in 4 rabbits. Twelve weeks after aneurysm creation, RNA was isolated from the aneurysm as well as the contralateral common carotid artery and used for microarray experiments. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on 1 animal as a confirmatory test. RESULTS: Ninety-six (46%) of 209 genes in the microarray were differentially expressed in the rabbit aneurysm compared with the contralateral common carotid artery. In general, differential gene expression followed specific molecular pathways. Similarities were found between rabbit aneurysms and human intracranial aneurysms, including increased metalloproteinase activity and decreased production of the extracellular matrix. RT-PCR results confirmed the differential expression found by the gene chip microarray. CONCLUSIONS: The molecular characteristics of the rabbit elastase-induced saccular aneurysm are described. The rabbit aneurysm model shares some molecular features with human intracranial aneurysms. Future studies can use the rabbit model and the new rabbit gene chip microarray to study the molecular aspects of saccular aneurysms.

Ribosomal DNA sequencing as a tool for identification of bacterial pathogens.

Conventional methods for the identification and characterization of clinical isolates of bacterial pathogens sometimes fall short when such isolates exhibit unusual phenotypic profiles. Recent advances in DNA sequencing technology have greatly enhanced the ability of the microbiologist to determine the identity of a bacterial isolate. Given the relative objectivity of DNA sequence information and growing availability of sequence information databases, a significant movement is now afoot to use molecular methods for the identification of clinical pathogens.

Two geographically distinct isolates of Borrelia burgdorferi from the United States share a common unique ancestor.

The genetic diversity of Borrelia burgdorferi isolates from several geographic regions was evaluated by nucleotide sequence analysis of the genes encoding 23S ribosomal RNA and outer surface protein A. Comparison of nucleotide sequences spanning 738 bp of the 23S ribosomal DNA from two unusual isolates, DN127 (Del Norte County, California) and 25015 (Millbrook, New York), to homologous sequences from other B. burgdorferi isolates from the United States and Russia identified several nucleotide sequence polymorphisms that are unique to these two isolates. Sequence analysis of a 615 nucleotide segment of the gene encoding outer surface protein A also revealed greater similarity of strains DN127 and 25015 (94.1%) compared to other US and Eurasian isolates. These data were further corroborated by genomic macrorestriction analysis, in which DN127 and 25015 demonstrated unique restriction digestion patterns. Our findings suggest that substantial genetic diversity of B. burgdorferi, rivaling that of European strains, exists among isolates from the United States. Strains DN127 and 25015 are unique among all B. burgdorferi isolates tested to date, and though isolated from opposite longitudinal extremes of the North American continent, are closely related.

Hepatitis C virus genotypes and hepatitis G virus in hemodialysis patients from Syria: identification of two novel hepatitis C virus subtypes.

High prevalence of hepatitis C (HCV) and hepatitis G (HGV) viruses has been reported among hemodialysis patients with substantial heterogeneity of HCV genotypes throughout the world. We studied HCV prevalence, clinical significance, genotype distribution, and HGV coinfection in hemodialysis patients from Syria. Ninety (75%) of 120 screened patients were HCV antibody positive. Forty-nine (87.5%) of 56 HCV antibody-positive patients had HCV RNA detected by the polymerase chain reaction. The HCV genotyping was possible in 37 of 49 patients (76%). The HCV genotype distribution was genotype 1a, seven (19%); genotype 1b, 10 (27%); genotype 4a, 11 (30%); unmatched sequences, nine (24%). Phylogenetic analysis of unmatched sequences indicated that they represent two distinct and novel subtypes of HCV genotype 4. Hepatitis G virus RNA was detected in 29 (59%) of the HCV RNA-positive patients. No differences were identified between patients infected with HCV alone and those coinfected with HGV. These data demonstrate that HCV infection is common in this population with a genotype distribution predominantly made up of types 1 and 4. Coinfection with HGV had no effect on the outcome of HCV infection.

Characterization of culture-derived spiral bacteria by 16S ribosomal RNA gene sequence analysis.

Broad range amplification and sequence analysis of the 16S ribosomal RNA gene was used to identify three spiral-form organisms. The agents were identified as Campylobacter fetus, "Flexispira rappini", and Borrelia burgdorferi, respectively, using either proprietary or public sequence databases. In each case, the rDNA sequence showed 99-100% homology with known sequence data. Sequence-based analysis for each isolate required only 2-3 days, whereas traditional means of identification took 8-12 days to complete. The identification of spirochetes and vibrio-like agents from human clinical samples is often time consuming and results may be difficult to interpret, sometimes due to atypical phenotypic characteristics. Analysis of 16S rDNA or other molecular targets may provide a way to accurately and rapidly characterize isolates that are recalcitrant to speciation.

Intra-articular decorin influences the fibrosis genetic expression profile in a rabbit model of joint contracture.

OBJECTIVES: The goal of this study was to determine whether intra-articular administration of the potentially anti-fibrotic agent decorin influences the expression of genes involved in the fibrotic cascade, and ultimately leads to less contracture, in an animal model. METHODS: A total of 18 rabbits underwent an operation on their right knees to form contractures. Six limbs in group 1 received four intra-articular injections of decorin; six limbs in group 2 received four intra-articular injections of bovine serum albumin (BSA) over eight days; six limbs in group 3 received no injections. The contracted limbs of rabbits in group 1 were biomechanically and genetically compared with the contracted limbs of rabbits in groups 2 and 3, with the use of a calibrated joint measuring device and custom microarray, respectively. RESULTS: There was no statistical difference in the flexion contracture angles between those limbs that received intra-articular decorin versus those that received intra-articular BSA (66° vs 69°; p = 0.41). Likewise, there was no statistical difference between those limbs that received intra-articular decorin versus those who had no injection (66° vs 72°; p = 0.27). When compared with BSA, decorin led to a statistically significant increase in the mRNA expression of 12 genes (p < 0.01). In addition, there was a statistical change in the mRNA expression of three genes, when compared with those without injection. CONCLUSIONS: In this model, when administered intra-articularly at eight weeks, 2 mg of decorin had no significant effect on joint contractures. However, our genetic analysis revealed a significant alteration in several fibrotic genes. Cite this article: Bone Joint Res 2014;3:82-8.

ASCL1 and RET expression defines a clinically relevant subgroup of lung adenocarcinoma characterized by neuroendocrine differentiation.

ASCL1 is an important regulatory transcription factor in pulmonary neuroendocrine (NE) cell development, but its value as a biomarker of NE differentiation in lung adenocarcinoma (AD) and as a potential prognostic biomarker remains unclear. We examined ASCL1 expression in lung cancer samples of varied histologic subtype, clinical outcome and smoking status and compared with expression of traditional NE markers. ASCL1 mRNA expression was found almost exclusively in smokers with AD, in contrast to non-smokers and other lung cancer subtypes. ASCL1 protein expression by immunohistochemical (IHC) analysis correlated best with synaptophysin compared with chromogranin and CD56/NCAM. Analysis of a compendium of 367 microarray-based gene expression profiles in stage I lung adenocarcinomas identified significantly higher expression levels of the RET oncogene in ASCL1-positive tumors (ASCL1(+)) compared with ASCL1(-) tumors (q-value <10(-9)). High levels of RET expression in ASCL1(+) but not in ASCL1(-) tumors was associated with significantly shorter overall survival (OS) in stage 1 (P=0.007) and in all AD (P=0.037). RET protein expression by IHC had an association with OS in the context of ASCL1 expression. In silico gene set analysis and in vitro experiments by ASCL1 shRNA in AD cells with high endogenous expression of ASCL1 and RET implicated ASCL1 as a potential upstream regulator of the RET oncogene. Also, silencing ASCL1 in AD cells markedly reduced cell growth and motility. These results suggest that ASCL1 and RET expression defines a clinically relevant subgroup of ∼10% of AD characterized by NE differentiation.

Detection of the Staphylococcal mecA gene by chemiluminescent DNA hybridization.

A new solution-phase DNA hybridization capture assay for the rapid detection of the mecA gene in clinical isolates of Staphylococcus was compared with multiplex PCR and disk diffusion methods. The assay uses a DNA capture probe immobilized on paramagnetic particles and a second DNA probe labeled with an acridinium ester. Bacteria from 24-h cultures are lysed, and the lysates are hybridized with the DNA probes. After magnetic separation to remove unhybridized labeled probe, the mecA gene is detected by the chemiluminescence of the hybridized probe. Four hundred consecutive staphylococcal isolates were assayed, including 147 S. aureus and 253 coagulase-negative Staphylococcus isolates. Among the S. aureus isolates, 14 of 147 were MecA+ by both the hybridization capture assay and PCR; 133 of 147 were MecA negative by both assays (positive and negative predictive values, 100%). Comparison of disk diffusion results with those obtained by genotypic methods indicated that 14 of 147 S. aureus isolates judged to be resistant were positive by both methods; 119 of 147 were Oxs and negative by both genotypic methods (positive and negative predictive values, 50 and 100%, respectively). The remaining 14 S. aureus isolates were MecA- Oxr; among these, 13 were beta-lactamase hyperproducers. For coagulase-negative staphylococci, 130 of 253 were MecA+ by the hybridization capture assay; 129 of 130 of these isolates were positive by PCR (positive and negative predictive values, 99.2 and 100%, respectively). Comparison with the disk diffusion assay showed that 128 of the coagulase-negative MecA+ isolates were Oxr; 111 of 253 were MecA- and Oxs (positive and negative predictive values, 90.8 and 99.1%, respectively). Thirteen coagulase-negative isolates were MecA-Oxr; among these, three were beta-lactamase hyperproducers. Comparison of the hybridization capture assay results with PCR indicates that the DNA hybridization assay is a sensitive and specific test for the detection of the mecA gene in clinical isolates of Staphylococcus.

Heritable susceptibility to severe Borrelia burgdorferi-induced arthritis is dominant and is associated with persistence of large numbers of spirochetes in tissues.

In human Lyme disease, symptoms with widely varying levels of severity have been observed. A mouse model of Lyme disease has been developed which allows analysis of mice with mild, moderate, and severe pathologies after inoculation with the spirochete Borrelia burgdorferi. To determine whether the differences in symptoms reflect differences in the number of spirochetes persisting in affected tissues, a sensitive PCR technique was developed to detect B. burgdorferi DNA in virtually any tissue of an infected mouse. This analysis, which detects DNA from as few as three spirochetes, revealed the presence of B. burgdorferi DNA in many tissues from severely arthritic C3H/HeJ mice as early as 1 week postinfection. The heart, ear, and ankle were particularly heavily infected, although B. burgdorferi DNA was also detected in spleen, liver, brain, kidney, bladder, uterus, and lymph nodes. In contrast, much lower levels of spirochete DNA were detected in tissues of infected BALB/c mice, which develop less severe arthritis when infected with B. burgdorferi than do C3H/HeJ mice. This difference was evident throughout the 5-week analysis. A competitive PCR method allowed determination of the absolute number of spirochete gene sequences in infected tissues. Ankles and hearts from C3H/HeJ mice were found to harbor 10(7) copies of the B. burgdorferi ospA gene, while these tissues from BALB/c mice contained 5- and 10-fold less B. burgdorferi DNA, respectively. The genetic regulation of severe pathology was analyzed by infecting the offspring of a cross between C3H/HeJ and BALB/c mice. The F1 mice developed severe arthritis and contained high levels of Borrelia DNA in the heart and ankle, similar to the C3H/HeJ parent. These findings indicate that susceptibility to severe arthritis is a dominant trait and suggest that it may correlate with high levels of persisting spirochetes. Models of pathology in Lyme disease should take into consideration the fact that severity of pathology may be directly related to the number of organisms in infected tissues.

Borrelia burgdorferi enzyme-linked immunosorbent assay for discrimination of OspA vaccination from spirochete infection.

Recombinant Lyme disease vaccines based on purified preparations of outer surface protein A (OspA) have been shown to be effective in preventing transmission of Borrelia burgdorferi in experimental animal models and are now being tested in humans. Since the most widely used screening tests for Lyme disease are based on a whole-cell sonicate of B. burgdorferi, serologic false positivity in vaccinated persons could result from reactivity to OspA within the antigen preparation. In order to avoid serologic false positivity in vaccinated subjects, we developed an immunoassay based on a low-passage-number, naturally occurring variant of B. burgdorferi which lacks the plasmid encoding OspA and OspB. The use of an antigen preparation derived from this organism provided sensitive and specific detection of B. burgdorferi seropositivity in experimental animals and in human Lyme disease cases. The OspA-B-negative enzyme-linked immunosorbent assay (ELISA) also appeared to be capable of discriminating the vaccinated state from vaccine failure and natural infection in experimental animals. Sera from human subjects participating in a vaccine trial gave false-positive results with an ELISA based on an OspA-containing strain, but no such reactivity was observed when the OspA-negative ELISA was used. We conclude that low-passage-number OspA-B-negative isolates in immunoassays may become useful for the immunologic discrimination of the vaccinated state, natural infection, and vaccine failure.

Perpetuation of the agent of human granulocytic ehrlichiosis in a deer tick-rodent cycle.

A human-derived strain of the agent of human granulocytic ehrlichiosis, a recently described emerging rickettsial disease, has been established by serial blood passage in mouse hosts. Larval deer ticks acquired infection by feeding upon such mice and efficiently transmitted the ehrlichiae after molting to nymphs, thereby demonstrating vector competence. The agent was detected by demonstrating Feulgen-positive inclusions in the salivary glands of the experimentally infected ticks and from field-derived adult deer ticks. White-footed mice from a field site infected laboratory-reared ticks with the agent of human granulocytic ehrlichiosis, suggesting that these rodents serve as reservoirs for ehrlichiae as well as for Lyme disease spirochetes and the piroplasm that causes human babesiosis. About 10% of host-seeking deer ticks were infected with ehrlichiae, and of these, 20% also contained spirochetes. Cotransmission of diverse pathogens by the aggressively human-biting deer tick may have a unique impact on public health in certain endemic sites.

Branched-DNA assay for detection of the mecA gene in oxacillin-resistant and oxacillin-sensitive staphylococci.

The identification of methicillin-resistant staphylococcus isolates in the clinical laboratory has typically been performed by using methods that detect phenotypic expression of resistance determinants. However, these methods may be difficult to interpret and some isolates do not express resistance until selective pressure is administered. Assays that detect genetic determinants are not subject to these limitations and have been effective in distinguishing isolates that are capable of expressing the resistance phenotype. In this study, a novel branched-DNA (bDNA) hybridization assay was used to test for the mecA gene in 416 clinical staphylococcal isolates. The results were compared with those obtained by a PCR-based assay and oxacillin disk diffusion. For 155 Staphylococcus aureus and 261 coagulase-negative Staphylococcus isolates, the bDNA assay and PCR results were 100% concordant. Among the S. aureus isolates, 20 were MecA+ and 135 were MecA-. For the coagulase-negative staphylococci, 150 were MecA+ and 111 were MecA-. The results from the genotypic detection methods were compared with those obtained by oxacillin disk diffusion. No discrepancies were detected among the S. aureus isolates; however, 10 coagulase-negative isolates were MecA+ but oxacillin sensitive and 1 isolate was MecA- but oxacillin resistant. Oxacillin resistance was induced in 6 of the 10 MecA+ isolates previously classified as oxacillin sensitive. These results suggest that the bDNA method described here is a sensitive and efficient method for detection of methicillin resistance in staphylococci and that genetic detection methods may be useful for detection of potential methicillin resistance in the clinical laboratory.

Bordetella holmesii-like organisms associated with septicemia, endocarditis, and respiratory failure.

We recovered an unusual bacterial strain from blood or sputum of three patients with septicemia, endocarditis, and/or respiratory failure. The three isolates were thin, curved, gram-negative, light brown, pigment-producing bacilli with variable catalase activity. They were asaccharolytic, oxidase-negative, nonmotile, and fastidious. Identification was not possible on the basis of these characteristics alone or in combination with cellular fatty acid profiles. Nucleic acid amplification and sequence analysis of the 16S rRNA gene revealed that all three isolates were identical and most closely related to the emerging pathogen Bordetella holmesii, diverging from the published sequence at three nucleotide positions (99.8% similarity). Isolation of a B. holmesii-like pathogen from sputum suggests that, in addition to producing septicemia, the organism may inhabit the respiratory tract like other Bordetella species.

Prevalence of granulocytic Ehrlichia infection among white-tailed deer in Wisconsin.

Human granulocytic ehrlichiosis (HGE) is caused by an agent that is nearly indistinguishable from the veterinary pathogens Ehrlichia equi and Ehrlichia phagocytophila. The deer tick, Ixodes scapularis, is a vector of the HGE agent, and the white-tailed deer is the primary host for adult Ixodes ticks. We assessed the distribution of granulocytic Ehrlichia infection among deer living within (Wisconsin) and outside (western and southern Iowa) the geographic range of L. scapularis. Whole-blood samples were tested for HGE 16S ribosomal DNA (rDNA) by PCR, and E. equi antibody was detected by indirect immunofluorescence assay (IFA). Antibody titers of > or = 1:64 were defined as positive, and all positive samples were retested with a second lot of substrate antigen. E. equi antibody was present in 14 (8%) of 187 Wisconsin deer and 0 of 60 Iowa specimens (rate ratio undefined; P = 0.025). An additional 30 serum samples from Wisconsin deer were excluded because IFA results were discrepant between substrate lots. The reciprocal antibody titers ranged from 64 to 512 (geometric mean, 141) for positive samples. PCR results were positive for 27 (15%) of 181 Wisconsin deer. The prevalence of infection in northwestern Wisconsin deer was not significantly different from that in central Wisconsin deer, as determined by IFA and PCR. In two samples that were sequenced, the 16S rDNA was nearly identical to that of the granulocytic Ehrlichia species but distinct from that of Anaplasma marginale. The DNA sequences of the samples differed from the published sequences for E. equi, E. phagocytophila, and the HGE agent by 1 or 2 nucleotides (> or = 99.1% homology) at phylogenetically informative sites. Granulocytic Ehrlichia organisms in deer are widely distributed within the geographic range of L. scapularis in Wisconsin. Deer may serve as useful sentinels for areas where HGE transmission to humans may occur.

Ixodes dammini as a potential vector of human granulocytic ehrlichiosis.

Little is known about the epidemiology and mode of transmission of the agent of human granulocytic ehrlichiosis (HGE). Analyses of an engorged female Ixodes dammini tick removed from an HGE patient and 101 field-collected I. dammini and Dermacentor variabilis from three Wisconsin counties for Borrelia burgdorferi and Ehrlichia phagocytophila/Ehrlichia equi DNA revealed that the patient tick and 7 of 68 I. dammini ticks from Washburn County collected in 1982 and 1991 were positive for ehrlichial DNA; 10 ticks from the same collections were positive for B. burgdorferi. Two specimens (2.2%) were positive for both organisms. Serologic evidence for exposure to the agent of HGE or its relatives was detected in 3 of 25 Lyme disease patients from the upper Midwest. These data argue that I. dammini is a common vector for transmission of both Lyme disease and HGE.

Direct mecA detection from blood culture bottles by branched-DNA signal amplification.

A branched-DNA (bDNA) signal amplification method was used to detect the mecA gene directly from blood culture broth growing staphylococci. BACTEC blood culture bottles with positive growth indices and containing staphylococcus-like organisms as shown by Gram stain were tested for the presence of the mecA gene. Comparison of test results was done among 225 patients (one blood culture from each patient). Compared with PCR, the sensitivity and specificity of the bDNA method are 100 and 99%, respectively. The bDNA test is carried out in a 96-well format and requires approximately 6 h to perform. Our preliminary results suggest that direct detection of the mecA gene by bDNA signal amplification is (i) sensitive enough to detect mecA directly from blood culture bottles without the requirement for subculture and (ii) as sensitive and specific as the PCR-based method.

Clinical significance of TT virus infection in patients with chronic hepatitis C.

OBJECTIVE: The TT virus (TTV) is a novel DNA virus that has recently been identified. The clinical significance of TTV infection in patients with chronic hepatitis C has not been determined. The aim of this study was to determine the prevalence and possible role of TTV in a well characterized population with chronic hepatitis C infection. METHODS: Ninety patients with chronic HCV and known time of HCV acquisition were selected from approximately 250 patients followed at our institution. Characteristics including age, sex, histology, and length of disease were recorded. Direct sequencing of the NS5 region was used for HCV genotyping. TTV DNA detection was based on PCR. RESULTS: TTV infection was present in 24 of 90 (27%) HCV patients. Patients were divided into four groups based on stage of disease; chronic hepatitis (CH, 29 patients), compensated cirrhosis (CC, 17 patients), decompensated cirrhosis (DC, 28 patients), and hepatocellular carcinoma (HCC, 16 patients). TTV was present in 2/29 (7%), 2/17 (12%), 11/28 (39%), and 9/16 (56%) in those with CAH, CC, DC, and HCC respectively. TTV was significantly more prevalent among those with advanced disease (DC and HCC) compared to those with stable disease (CH and CC; p = 0.001). Mean age, sex, and the time from exposure to HCV to development of complications were similar in TTV-positive and -negative patients. TTV infection was more common in patients infected with HCV genotype 1b. Univariate analysis showed that length of HCV infection, HCV genotype 1b, and TTV infection were important in predicting the stage of liver disease in HCV patients. However, after adjusting for length of HCV infection, TTV but not HCV genotype was important in predicting the stage of liver disease. CONCLUSIONS: We conclude that 1) TTV infection is common in patients with chronic HCV; 2) TTV infection is more prevalent among patients with advanced HCV-associated liver disease (DC and HCC) than in those with stable disease (CH and CC); and 3) TTV infection is more common in patients with HCV genotype 1b but is independent from genotype in predicting the stage of HCV-associated liver disease.

Serodiagnosis of human granulocytic ehrlichiosis by using novel combinations of immunoreactive recombinant proteins.

A panel of seven recombinant antigens, derived from Ehrlichia phagocytophila (the agent of human granulocytic ehrlichiosis), was evaluated by class-specific enzyme-linked immunosorbent assays (ELISAs) for utility in the diagnosis of the infection. Fourteen genomic fragments, obtained by serologic expression screening, contained open reading frames (ORFs) encoding 16 immunodominant antigens. Eleven of these antigens were members of the major surface protein (MSP) multigene family. Alignment of their predicted protein sequences revealed a pattern of conserved sequences, which contained short direct repeats, flanking a variable region. In addition, two genomic clones contained two and three MSP ORFs, respectively, indicating that these genes are clustered in tandem copies. The implications for this pattern of both genomic and protein arrangements in antigenic variations of MSPs and in their utilities in a diagnostic assay are discussed. In addition to two MSP recombinant antigens (rHGE-1 and -3) and a fusion protein of these antigens (rErf-1), five further recombinants were evaluated by ELISA. Two of these antigens (rHGE-14 and -15) were novel, while a third (rHGE-2), with no known function, has been described. The final two recombinant antigens (rHGE-9 and -17) represent overlapping segments of the ankyrin gene (ank). The addition of rHGE-9 ELISA data resulted in the detection of 78% (21 of 27) of acute-phase sera. When serologic data for all recombinants are combined, 96.2% (26 of 27) of convalescent-phase patient serum samples and 85.2% (23 of 27) of acute-phase patient serum samples are detected, indicating the potential of these antigens for use in the development of a rapid serologic assay for the detection of E. phagocytophila infection.

Characterization of an immunoreactive protein from the agent of human granulocytic ehrlichiosis.

A gene that is homologous to the Ehrlichia chaffeensis groEL operon was recovered and characterized by broad-range PCR amplification of whole blood from patients with human granulocytic ehrlichiosis (HGE) and from infected HL60 cell cultures. Sequence analysis of an 820-bp DNA fragment recovered directly from human blood showed 76.5 and 76.3% identity with cognate sequences from E. chaffeensis and Cowdria ruminantium, respectively. Analysis of a 1.6-kb DNA fragment derived from an HGE agent-infected HL60 cell culture indicated a near-complete open reading frame that contained 75.6 and 75.2% sequence identity with the E. chaffeensis and C. ruminantium groEL sequences, respectively. Phylogenetic analysis of this fragment showed that the HGE agent-derived sequence was related to, but distinct from, the sequences of E. chaffeensis and C. ruminantium. Polyvalent antibody responses to a recombinant fusion protein based on the HGE agent groEL homolog were detected in three of three BALB/c mice that were infected by syringe inoculation with a Wisconsin strain of the HGE agent (WI-1) and nine of nine mice infected by Ixodes scapularis (Ixodes dammini) tick inoculation of an isolate from Nantucket Island, Mass. (NCH-1). No response was detected in mice infected with Borrelia burgdorferi or in control BALB/c mice. Further characterization of the sensitivity and specificity of immune responses to this protein will be facilitated by the use of recombinant fusion proteins or peptides based on the HGE agent-specific groEL homolog.