Genetic diversity of Ehrlichia canis in Brazil.

Canine monocytic ehrlichiosis is a highly prevalent disease in Brazil, where the genetic diversity of Ehrlichia canis remains undefined. In this study, we used the TRP36 gene to examine the genetic diversity of E. canis strains from naturally infected dogs residing in five distinct geographic regions in Brazil. E. canis DNA was detected in 82/126 (65%) dogs by dsb-specific PCR and E. canis was isolated in cell culture from 13 dogs. Sequences obtained from dsb genes amplified from the isolates were identical to the US E. canis strain. An extended molecular characterization based on the TRP36 gene identified two major genogroups based on differences among eight isolates. Isolates with tandem repeat amino acid sequence (TEDSVSAPA) identical to the previously reported TRP36 sequence were found in the midwest, northeast and southeast regions of Brazil, and classified into the US genogroup. A novel Brazilian genotype with a different tandem repeat sequence (ASVVPEAE) was also identified in midwest, northern and southern regions. Similarity in the N-terminal sequence of a US genogroup member with the Brazilian genogroup suggested that genomic recombination between the two genogroups may have occurred. Other subtypes within the Brazilian genogroup were also identified using C-terminal amino acid divergence. We identified two distinct major Brazilian genogroups and several subtypes based on analysis of TRP36, and such information will be useful for further genotyping and possible associations with disease severity, understanding of the genetic and antigenic variability of E. canis, and for developing strain-specific vaccines and diagnostic methods based on TRP36.

Optical fiber imaging for high speed plasma motion diagnostics: applied to low voltage circuit breakers.

An integrated portable measurement system is described for the study of high speed and high temperature unsteady plasma flows such as those found in the vicinity of high current switching arcs. An array of optical fibers allows the formation of low spatial resolution images, with a maximum capture rate of 1 x 10(6) images per second (1 MHz), with 8 bit intensity resolution. Novel software techniques are reported to allow imaging of the arc; and to measure arc trajectories. Results are presented on high current (2 kA) discharge events in a model test fixture and on the application to a commercial low voltage circuit breaker.

Molecular and clinical evidence of Ehrlichia chaffeensis infection in Cameroonian patients with undifferentiated febrile illness.

In the U.S.A., human monocytotropic ehrlichiosis (HME) caused by Ehrlichia chaffeensis is an emerging tick-transmitted zoonosis. In Cameroon, where E. canis, E. chaffeensis and E. ewingii have recently been detected in dogs and/or ticks (Rhipicephalus sanguineus), the potential exists for human infections. Patients from the coastal region of Cameroon who had acute fevers of unknown aetiology were therefore checked for ehrlichial infection, using a real-time PCR that amplifies part of a genus-specific gene (dsb) that codes for a disulphide-bond formation protein. Ehrlichial blood was detected in the peripheral blood from 12 (10%) of the 118 patients investigated by PCR. When the 12 amplicons from the positive cases were sequenced, they were found to be identical to each other and to the corresponding dsb sequence of an Arkansas strain of E. chaffeensis. The 12 patients who were PCR-positive for E. chaffeensis suffered from fever (100%), headache (67%), myalgia (42%), arthralgia (58%), pulmonary involvement (17%) and/or a diffuse rash (17%).

The genome of the obligately intracellular bacterium Ehrlichia canis reveals themes of complex membrane structure and immune evasion strategies.

Ehrlichia canis, a small obligately intracellular, tick-transmitted, gram-negative, alpha-proteobacterium, is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis. Complete genome sequencing revealed that the E. canis genome consists of a single circular chromosome of 1,315,030 bp predicted to encode 925 proteins, 40 stable RNA species, 17 putative pseudogenes, and a substantial proportion of noncoding sequence (27%). Interesting genome features include a large set of proteins with transmembrane helices and/or signal sequences and a unique serine-threonine bias associated with the potential for O glycosylation that was prominent in proteins associated with pathogen-host interactions. Furthermore, two paralogous protein families associated with immune evasion were identified, one of which contains poly(G-C) tracts, suggesting that they may play a role in phase variation and facilitation of persistent infections. Genes associated with pathogen-host interactions were identified, including a small group encoding proteins (n = 12) with tandem repeats and another group encoding proteins with eukaryote-like ankyrin domains (n = 7).

Ehrlichial infection in Cameroonian canines by Ehrlichia canis and Ehrlichia ewingii.

Ehrlichia chaffeensis and Ehrlichia ewingii are agents of emerging human ehrlichioses in North America and are transmitted primarily by Amblyomma americanum ticks, while Ehrlichia canis is the globally distributed cause of canine monocytic ehrlichiosis (CME) and is transmitted by the brown dog tick, Rhipicephalus sanguineus. Although E. canis and Ehrlichia ruminantium are endemic in Africa, the presence of ehrlichial agents in dogs and ticks in Cameroon has not been investigated. The objective of this study was to determine the prevalence of ehrlichial infections in Cameronian dogs using a combination of serologic and molecular methods. Peripheral blood was collected, clinical signs and the presence or absence of ticks on dogs (n=104) presenting for various reasons at local veterinary clinics around the Mount Cameroon region were noted. IFA identified 33 dogs (32%) with antibodies reactive with E. canis, and reactivity of these sera with all major E. canis antigens (200, 140, 95, 75, 47, 36, 28, and 19-kDa) was confirmed by immunoblotting. Multicolor real-time PCR detected ehrlichial DNA (E. canis (15) and E. ewingii (2)) in 17 dogs (16.3%), all of which had attached ticks at time of presentation. The dsb amplicons (378 bp) from E. canis and E. ewingii were identical to gene sequences from North American isolates. This study identifies canine ehrlichiosis as a prevalent unrecognized cause of disease in Cameroonian canines.

Phylogenetic relationships of Anaplasma marginale and 'Ehrlichia platys' to other Ehrlichia species determined by GroEL amino acid sequences.

The heat-shock protein (GroEL) genes of Anaplasma marginale, Ehrlichia muris and 'Ehrlichia platys' were sequenced and compared with the GroEL of other species of Ehrlichia. The GroEL amino acid sequences of A. marginale and 'E. platys' were most similar to the GroEL sequence of Ehrlichia phagocytophila, with which they formed one group with 6-10% divergence. The E. muris GroEL was most closely related to the GroEL of two unclassified strains (HF-565 and Anan), then to Ehrlichia chaffeensis, Ehrlichia canis, Ehrlichia ewingii and Cowdria ruminantium, forming a second distinct group (0.3-8.6% divergence). The GroELs of Ehrlichia risticii and Ehrlichia sennetsu were very similar to one another (only 2% divergence), forming the third group. The first two groups were relatively closely related (17-20% divergence), while the third group was only distantly related to the first two groups (62-73% divergence).

Immunodiagnosis of Ehrlichia canis infection with recombinant proteins.

Ehrlichia canis causes a potentially fatal rickettsial disease of dogs that requires rapid and accurate diagnosis in order to initiate appropriate therapy leading to a favorable prognosis. We recently reported the cloning of two immunoreactive E. canis proteins, P28 and P140, that were applicable for serodiagnosis of the disease. In the present study we cloned a new immunoreactive E. canis surface protein gene of 1,170 bp, which encodes a protein with a predicted molecular mass of 42.6 kDa (P43). The P43 gene was not detected in E. chaffeensis DNA by Southern blot, and antisera against recombinant P43 (rP43) did not react with E. chaffeensis as detected by indirect fluorescent antibody (IFA) assay. Forty-two dogs exhibiting signs and/or hematologic abnormalities associated with canine ehrlichiosis were tested by IFA assay and by recombinant Western immunoblot. Among the 22 samples that were IFA positive for E. canis, 100% reacted with rP43, 96% reacted with rP28, and 96% reacted with rP140. The specificity of the recombinant proteins compared to the IFAs was 96% for rP28, 88% for P43 and 63% for P140. The results of this study demonstrate that the rP43 and rP28 are sensitive and reliable serodiagnostic antigens for E. canis infections.

A conserved, transcriptionally active p28 multigene locus of Ehrlichia canis.

Antigenic diversity of Ehrlichia chaffeensis and Ehrlichia canis may involve independent or differential expression of the P28 outer membrane proteins genes, enabling persistent infections of the natural hosts. In this study, we analyzed the transcriptional activity of a five gene locus in E. canis encoding homologous, but non-identical, p28 genes. The p28 multigene locus contained three previously identified complete gene sequences and one partial gene sequence. A new p28 gene was identified and sequenced, and the complete sequence of a second partial p28 gene was determined. The new p28 gene joined two previously separate loci, forming the single p28 multigene locus. The amino acid homology of the E. canis P28 proteins ranged from 51 to 74%. The nucleic acid sequence of regions compared within the locus spanning four p28 genes from two geographically distinct E. canis isolates was completely conserved. Analysis of the five p28 genes demonstrated that all were transcriptionally active in in-vitro cultures of E. canis incubated at the vertebrate host (37 degrees C) and ambient tick temperatures (27 degrees C). Polycistronic copies of multiple p28 genes were not detected by RT-PCR, but monocistronic p28 mRNA transcripts were detected by Northern blotting from E. canis infected DH82 cells, indicating that the genes are transcribed as monocistronic messages.

Characterization of the complete transcriptionally active ehrlichia chaffeensis 28 kDa outer membrane protein multigene family.

The 28kDa outer membrane proteins (P28) of Ehrlichia chaffeensis are encoded by a multigene family. The purpose of this study was to determine all the p28 gene sequences and their transcriptional activities. There were 21 members of the p28 multigene family located in a 23kb DNA fragment in the genome of E. chaffeensis. The p28 genes each contained 816-903 nucleotides with intergenic spaces of 10-605 nucleotides. All the genes were complete and were predicted to have a signal sequence. The molecular masses of the mature proteins were predicted to be 28-32kDa. The amino acid sequence identity of the P28 proteins was 20-83%. Ten p28 genes were investigated for transcriptional activity by using RT-PCR amplification of mRNA. Six of 10 tested p28 genes were actively transcribed in cell-culture grown E. chaffeensis. RT-PCR also indicated that each of the p28 genes was monocistronic. These results suggest that the p28 genes are active genes and encode polymorphic forms of the P28 proteins. The P28s were divergent among isolates of E. chaffeensis also. The large repertoire of the p28 genes in a single ehrlichial organism and antigenic diversity of the P28 among the isolates of E. chaffeensis suggest that P28s may be involved in immune avoidance.

Molecular cloning and characterization of the 120-kilodalton protein gene of Ehrlichia canis and application of the recombinant 120-kilodalton protein for serodiagnosis of canine ehrlichiosis.

The 120-kDa outer membrane protein (p120) is a potential adhesin of Ehrlichia chaffeensis, and recombinant p120 is very useful for serodiagnosis of human monocytotropic ehrlichiosis. The analogous gene of p120 in Ehrlichia canis was cloned, sequenced, and expressed. Like the E. chaffeensis p120, the E. canis p120 contains tandem repeat units. However, neither the repeat number nor the amino acid sequences in the repeats are identical in the two Ehrlichia species. The repeat units are hydrophilic and by probability analysis are predicted to be surface exposed in both species. The repeat regions of the p120s of the two species have common amino acid sequences that are predicted to be surface exposed. The overall amino acid sequence of the E. canis p120 is 30% homologous to that of E. chaffeensis p120. Protein immunoblotting demonstrated that the recombinant E. canis p120 reacted with convalescent sera from dogs with canine ehrlichiosis. These results indicate that the recombinant p120 is a potential antigen for the serodiagnosis of canine ehrlichiosis.

Glycosylation of homologous immunodominant proteins of Ehrlichia chaffeensis and Ehrlichia canis.

The glycoprotein genes of Ehrlichia chaffeensis (1,644 bp) and Ehrlichia canis (2,064 bp) encode proteins of 548 to 688 amino acids with predicted molecular masses of only 61 and 73 kDa but with electrophoretic mobilities of 120 kDa (P120) and 140 kDa (P140), respectively. The 120-kDa protein gene of E. chaffeensis contains four identical 240-bp tandem repeat units, and the 140-kDa protein gene of E. canis has 14 nearly identical, tandemly arranged 108-bp repeat units. Conserved serine-rich motifs identified in the repeat units of P120 and P140 were also found in the repeat units of the human granulocytotropic ehrlichiosis agent 130-kDa protein and of the fimbria-associated adhesin protein Fap1 of Streptococcus parasanguis. Nearly the entire (99%) E. chaffeensis P120 gene (1,616 bp), the 14-repeat region (78%) of the E. canis P140 gene (1,620 bp), and a 2-repeat region from the E. chaffeensis P120 gene (520 bp) were expressed in Escherichia coli. The recombinant proteins exhibited molecular masses ranging from 1.6 to 2 times larger than those predicted by the amino acid sequences. Antibodies against the recombinant proteins reacted with E. chaffeensis P120 and E. canis P140, respectively. Carbohydrate was detected on the E. chaffeensis and E. canis recombinant proteins, including the two-repeat polypeptide region of E. chaffeensis P120. A carbohydrate compositional analysis identified glucose, galactose, and xylose on the recombinant proteins. The presence of only one site for N-linked (Asn-Xaa-Ser/Thr) glycosylation, a lack of effect of N-glycosidase F, the presence of 70 and 126 Ser/Thr glycosylation sites in the repeat regions of P120 and P140, respectively, and a high molar ratio of carbohydrate to protein suggest that the glycans may be O linked.

Molecular cloning of the gene for a conserved major immunoreactive 28-kilodalton protein of Ehrlichia canis: a potential serodiagnostic antigen.

A gene encoding a 28-kDa protein of Ehrlichia canis was cloned, sequenced, and expressed, and a comparative molecular analysis with homologous genes of E. canis, Cowdria ruminantium, and Ehrlichia chaffeensis was performed. The complete gene has an 834-bp open reading frame encoding a protein of 278 amino acids with a predicted molecular mass of 30.5 kDa. An N-terminal signal sequence was identified, suggesting that the protein undergoes posttranslational modification to a mature 27.7-kDa protein (P28). The E. canis p28 gene has significant nucleic acid and amino acid sequence homologies with the E. chaffeensis outer membrane protein-1 (omp-1) gene family, with the Cowdria ruminantium map-1 gene, and with other E. canis 28-kDa-protein genes. Southern blotting revealed the presence of at least two additional homologous p28 gene copies in the E. canis genome, confirming that p28 is a member of a polymorphic multiple-gene family. Amino acid sequence analysis revealed that E. canis P28 has four variable regions, and it shares similar surface-exposed regions, antigenicity, and T-cell motifs with E. chaffeensis P28. The p28 genes from seven different E. canis isolates were identical, indicating that the gene for this major immunoreactive protein is highly conserved. In addition, reactivity of sera from clinical cases of canine ehrlichiosis with the recombinant P28 demonstrated that the recombinant protein may be a reliable serodiagnostic antigen.

Evidence of Pasteurella haemolytica linked immune complex disease in natural and experimental models.

The pathogenesis of bovine pneumonic pasteurellosis is not completely understood, and studies have not established that Pasteurella haemolytica A1 (Ph1) virulence is exclusively responsible for the development of acute pulmonary lesions. The purpose of this investigation was to determine if immune complex disease is involved in the pathogenesis of bovine pneumonic pasteurellosis. A retrospective immunohistologic study of lung tissue from natural cases of bovine pneumonic pasteurellosis (44) as performed, and immune complexes were observed in alveloar spaces and walls in 88% of these cases. To study this pathologic mechanism experimentally, groups of mice were immunized with purified Ph1 outer membranes (OMs) or sham immunized on days 0 and 14. Mice were challenged intratracheally on day 24 with either live Ph1 or Ph1 OMs, and pulmonary lesions were assessed 24 h after challenge. Placebo immunized mice developed focal infiltrates of neutrophils and macrophages centered around large caliber bronchi. Mice immunized with Ph1 OMs and challenged with live Ph1 or OMs developed severe bronchointerstitial pneumonia with diffuse neutrophilic infiltration, focal necrosis, hemorrhage and edema, that is histologically similar to bovine pneumonic pasteurellosis. Immunohistology revealed flocculent aggregates of IgG and complement positive material within alveolar spaces and walls from mice challenged with live Ph1, and fine granular deposits of IgG and complement positive material were observed lining the alveolar walls from mice challenged with Ph1 OMs. Immunized mice exhibited high serum IgG antibody titers to Ph1 outer membrane proteins (OMPs). Results of this study suggest that immune complex disease plays a role in the pathogenesis of bovine pneumonic pasteurellosis.

Primary and anamnestic responses of bovine bronchoalveolar and peripheral blood lymphocyte subsets to aerosolized Pasteurella haemolytica A1.

Site-specific responses of bronchoalveolar and peripheral blood lymphocyte subsets were compared during primary and anamnestic immune responses against live Pasteurella haemolytica A1 (Ph1). Eight 1-year old calves were sequentially exposed intrabronchially with aerosolized Ph1 on days 0, 14, and 21, and two calves were sham exposed. Bronchoalveolar and peripheral blood lymphocytes were analyzed before each Ph1 exposure, and on days 3 and 7 post exposure using single and two-color flow cytometry to identify CD2+, CD4+, CD8+, CD21+, CD45R+, CD25+ and gammadelta lymphocyte subsets. Significant differences (p < 0.05) in bronchoalveolar and peripheral blood lymphocyte subsets were observed before Ph1 exposure. Subsequent aerosol exposures, resulted in significant (p < 0.05) changes in bronchoalveolar lymphocyte subsets and the CD4:CD8 bronchoalveolar lymphocyte ratio, but concomitant changes were not observed in peripheral blood lymphocytes. Expression of CD2, CD4 and CD8 lymphocyte differentiation antigens was consistently lower and more heterogeneous on bronchoalveolar lymphocytes. Differential analysis of bronchoalveolar leukocytes revealed a significant increase in bronchoalveolar lymphocytes and neutrophils during anamnestic responses.

Genetic diversity of the 28-kilodalton outer membrane protein gene in human isolates of Ehrlichia chaffeensis.

The Ehrlichia chaffeensis 28-kDa outer membrane protein (p28) gene was sequenced completely by genomic walking with adapter PCR. The DNA sequence of the p28 gene was nearly identical to the previously reported sequence (N. Ohashi, N. Zhi, Y. Zhang, and Y. Rikihisa, Infect. Immun. 66:132-139, 1998), but analysis of a further 75 bp on the 5' end of the gene revealed DNA that encoded a 25-amino-acid signal sequence. The leader sequence was removed from the N terminus of a 30-kDa precursor to generate the mature p28 protein. A monoclonal antibody (MAb), 1A9, recognizing four outer membrane proteins of E. chaffeensis (Arkansas strain) including the 25-, 26-, 27-, and 29-kDa proteins (X.-J. Yu, P. Brouqui, J. S. Dumler, and D. Raoult, J. Clin. Microbiol. 31:3284-3288, 1993) reacted with the recombinant p28 protein. This result indicated that the four proteins recognized by MAb 1A9 were encoded by the multiple genes of the 28-kDa protein family. DNA sequence alignment analysis revealed divergence of p28 among all five human isolates of E. chaffeensis. The E. chaffeensis strains could be divided into three genetic groups on the basis of the p28 gene. The first group consisted of the Sapulpa and St. Vincent strains. They had predicted amino acid sequences identical to each other. The second group contained strain 91HE17 and strain Jax, which only showed 0.4% divergence from each other. The third group contained the Arkansas strain only. The amino acid sequences of p28 differed by 11% between the first two groups, by 13.3% between the first and third groups, and by 13.1% between the second and third groups. The presence of antigenic variants of p28 among the strains of E. chaffeensis and the presence of multiple copies of heterogeneous genes suggest a possible mechanism by which E. chaffeensis might evade the host immune defenses. Whether or not immunization with the p28 of one strain of E. chaffeensis would confer cross-protection against other strains needs to be investigated.

Memory and CD8+ are the predominant bovine bronchoalveolar lymphocyte phenotypes.

Bovine lymphocytes obtained by bronchoalveolar lavage (BAL) of healthy calves were simultaneously analyzed and compared to peripheral blood lymphocytes using monoclonal antibodies specific for bovine leukocyte differentiation antigens. Phenotypic differences were observed between bronchoalveolar and peripheral blood T-lymphocyte subpopulations, demonstrating selective lymphocyte migration to the bovine lung. The bronchoalveolar and peripheral blood T-lymphocyte populations, defined by expression of CD2, were similar, but bronchoalveolar T lymphocytes were predominately CD8+ while peripheral blood T cells were predominately CD4+. In addition, memory lymphocytes, characterized by low expression of CD45R and activated lymphocytes (CD25+), were found in significantly higher proportions in the bronchoalveolar compartment. The proportion of gammadelta T lymphocytes was, however, significantly higher in peripheral blood. B cells were observed in similar proportions in the bronchoalveolar compartment and peripheral blood.

Diffuse intimal fibromuscular dysplasia with multiorgan failure.

A woman presented with a rapid onset of hypertension, angina pectoris, peripheral vascular disease, renal involvement, and a large liver cyst. Surgical removal of the liver cyst precipitated renal and liver failure and a terminal arrhythmia. At autopsy, there was intimal fibromuscular dysplasia involving the arteries to the heart, liver, kidneys, and intestines and evidence of recent infarction of the intestines, kidney, and liver. This case illustrates that intimal fibromuscular dysplasia (FMD) can be a diffuse and rapidly progressive disease. Some treatments currently being evaluated for preventing restenosis following angioplasty may find use in treating this uncommon disease.

PCR detection of acute Ehrlichia canis infection in dogs.

A polymerase chain reaction (PCR)-based detection assay that specifically detected Ehrlichia canis in dogs with acute infections was developed. A region of the 16S ribosomal RNA gene of E. canis was targeted for PCR amplification and chemiluminescent hybridization (CH) with a complementary internal 287-base pair (bp) oligonucleotide probe. The CH improved the PCR assay sensitivity 1,000-fold as compared with visualization on ethidium bromide-stained agarose gels. The PCR assay with CH (PCR/CH) detected as little as 30 fg of E. canis genomic DNA, the equivalent of approximately 150 E. canis organisms. The 495-bp product defined by the specific primers was not detected when genomic DNA from E. platys, E. chaffeensis, E. risticii, and E. equi were used in the PCR/CH assay. The PCR/CH assay was tested with unfractionated blood samples collected from 9 dogs experimentally infected with E. canis. The PCR/CH assay had greater detection sensitivity than did cell culture isolation (CCI) from infected blood. PCR/CH detected E. canis 7 days prior to CCI in 4 of 6 experimentally infected dogs. The results obtained with the PCR/CH assay otherwise consistently matched the results obtained by CCI. This PCR/CH assay is a rapid, sensitive, and specific method for E. canis detection with sensitivity comparable to or exceeding that of CCI. A diagnosis of E. canis using this PCR/CH assay can be made in 2 days as compared with 1-4 weeks for CCI. The PCR/CH assay appears to be an acceptable alternative or complement to current diagnostic techniques.

Characterization of bovine pulmonary and serum antibody responses after parenteral or intrapulmonary vaccination with live Pasteurella haemolytica.

Pulmonary and serum antibody responses were evaluated in eight calves vaccinated [four intrapulmonary-right diaphragmatic lobe (IP) and four subcutaneous (SC)] with Pasteurella haemolytica A1 (Ph-1) impregnated agar beads and eight respective sham-vaccinated calves. Experimental and sham groups were challenged in both diaphragmatic lobes with Ph-1 34-37 d after vaccination (DAV) and necropsied 6 d after challenge (DAC; 40-43 DAV). IgG antibodies contained in fluids from the diaphragmatic lobes of vaccinated calves had different patterns of antigen specificity compared with IgG antibodies in analogous sera. Using ELISA, anti-Ph-1 IgA and IgG antibody concentrations were significantly higher (P < 0.05) in lung lavage fluids from the IP group before and after challenge compared to the SC and sham groups. The IP and SC groups developed IgA, IgG and IgM antibody titers in nonvaccinated lung lobes after vaccination and challenge. The IP and SC groups exhibited significantly (P < 0.05) smaller pulmonary lesions than the sham groups and pulmonary IgG and IgA antibodies were associated with increased protection.

Development of polymerase chain reaction for specific identification of epizootic hemorrhagic disease virus serotype 1.

The diagnostic potential of the polymerase chain reaction (PCR) for specific identification of epizootic hemorrhagic disease virus serotype 1 (EHDV-1) in cell culture and clinical specimens was evaluated. Using oligonucleotide primers, selected from genome segment 2 of EHDV-1 (New Jersey strain), the PCR-based assay resulted in a 862 base pair (bp) PCR product. EHDV-1 RNA from United States prototype serotype 1 and a number of EHDV-1 field isolates, propagated in cell cultures, were detected by this PCR based assay. The specific 862 bp PCR products were visualized on ethidium bromide-stained agarose gel. Identity of the PCR product was confirmed by chemiluminescent hybridization with non radiolabelled internal probe. Using chemiluminescent hybridization, the sensitivity of the PCR assay was 1.0 fg of virus RNA (equivalent to 60 virus particles). Amplification product was not detected when the PCR-based assay was applied to RNA from EHDV serotype 2 (EHDV-2); the United States bluetongue virus (BLU) prototypes serotypes 2, 10, 11, 13, and 17; total nucleic acid extracts from uninfected BHK-21 cell; or blood cells from calves and deer that were EHDV-seronegative and virus isolation negative. Application of this EHDV-1 PCR-based assay to clinical samples resulted in detection of EHDV-1 RNA from blood samples, collected from a calf experimentally infected with EHDV-1. The described PCR-based assay provides a simple, rapid, sensitive, specific and inexpensive method for specific identification of EHDV-1 infection in susceptible ruminants.