Detection and identification of Ehrlichia species in blood by use of PCR and electrospray ionization mass spectrometry.

Rapid detection and identification of Ehrlichia species improves clinical outcome for patients suspected of ehrlichiosis. We describe an assay that employs multilocus PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) to detect and identify Ehrlichia species directly from blood specimens. The results were compared to those of a colorimetric microtiter PCR enzyme immunoassay (PCR-EIA) used as a diagnostic assay. Among 213 whole-blood samples collected from patients who were clinically suspected of ehrlichiosis from 1 May to 1 August 2008 at Vanderbilt University Hospital, 40 were positive for an Ehrlichia species by PCR/ESI-MS, giving a positive rate of 18.8%. In comparison to the PCR-EIA, PCR/ESI-MS possessed a sensitivity, a specificity, and positive and negative predictive values of 95.0%, 98.8%, 95.0%, and 98.8%, respectively. The 38 specimens that were positive for Ehrlichia by both PCR/ESI-MS and the PCR-EIA were further characterized to the species level, with 100% agreement between the two assays. In addition, Rickettsia rickettsii was detected by PCR/ESI-MS from four specimens that were confirmed retrospectively by serology and PCR-EIA. In three specimens, the PCR/ESI-MS assay identified Pseudomonas aeruginosa, Neisseria meningitidis, and Staphylococcus aureus; these were confirmed by culture and/or clinical diagnosis as being clinically relevant. From specimen processing to result reporting, the PCR/ESI-MS assay can be completed within 6 h, providing another laboratory tool for the diagnosis of ehrlichiosis. Moreover, this system may provide rapid detection and identification of additional pathogens directly from blood specimens.

Antigenic protein modifications in Ehrlichia.

To develop effective vaccination strategies against Ehrlichia, we have previously reported developing an animal model of cross-protection in which C57BL/6 mice primed with E. muris were resistant to lethal infection with Ixodes ovatus ehrlichia (IOE). Polyclonal antibody produced in mice after priming with E. muris and later injected with IOE-detected antigenic proteins in E. muris and IOE cell lysates. Cross-reaction of antigenic proteins was observed when we probed both the E. muris and IOE cell lysates with IOE and E. muris-specific polyclonal antibody. Analysis of the total proteins of E. muris and IOE by two dimensional electrophoresis showed that both E. muris and IOE have the same antigenic proteins. Finally, studies on post-translational protein modifications using a novel technique, Eastern blotting, showed that E. muris proteins are more lipoylated and glycosylated than those of IOE.

A simian model of human granulocytic ehrlichiosis.

Following intravenous inoculation with horse blood-infected with the agent of human granulocytic ehrlichiosis (HGE) from a human fatality, two rhesus macaques (Macaca mulatta) exhibited pyrexia and lethargy on days 4-12 postinfection (PI). Hematology revealed neutropenia, thrombocytopenia, and anemia, with ehrlichial morulae in monocytes and neutrophils on days 4-12. Blood was polymerase chain reaction (PCR)-positive on days 4-12 and bone marrow was PCR-positive on day 11. There was a minor increase in gamma-glutamyl transpeptidase on day 12 and serum interferon-gamma levels increased by day 18. Seroconversion occurred on day 20 PI to a titer of 100 by day 22. Western blot bands characteristic of HGE included 25-, 44-, 80-, 94-, 105-, and 125-kD bands. There was generalized lymphohistiocytic infiltration in the liver, spleen, lymph nodes, and other tissues. The liver had focal hepatocyte apoptosis. There was HGE DNA (by PCR) only in the spleen. Comparable findings were not observed in a monkey that received uninfected horse blood as a control. This animal model of human disease is important for further studies of HGE diagnosis, management, and pathogenesis.

Studies with recombinant proteins of Ehrlichia risticii: identification of strain-specific antigen as a protective antigen.

Ehrlichia risticii is the causative agent of Potomac horse fever, an acute infectious disease of equines. To study the role of major antigens of E. risticii in protective immune response, we have expressed the genes of the 55 kDa, 51 kDa and 85/50 kDa-strain-specific antigens of the 90-12 (85 kDa antigen) and 25-D (50 kDa antigen) strains in Escherichia coli using pRSET A, B, C system (Invitrogen, San Diego, CA). Mice immunized with these purified recombinant proteins of E. risticii developed strong and specific humoral immune response. The recombinant 85 kDa antigen of the 90-12 strain protected mice against challenge infection with both E. risticii strains, whereas its homologue from the 25-D strain, the recombinant 50 kDa antigen, protected mice against only the homologous strain challenge, but not against the heterologous 90-12 strain. Sera from mice immunized with the 85- or 50-kDa antigens did not inhibit the replication of cell-free Ehrlichiae in in vitro neutralization assays. Sera from normal mice and mice immunized with other antigens caused non-specific neutralization of E. risticii. Immunoglobulin G from mice immunized with the 51 kDa protein of the 90-12 strain caused partial in vitro neutralization of both strains of E. risticii. These studies demonstrate that the 85/50-kDa-strain-specific antigen of E. risticii is involved in immunoprotection against PHF.

Infection rate of Ehrlichia risticii, the agent of Potomac horse fever, in freshwater stream snails (Juga yrekaensis) from northern California.

Juga yrekaensis freshwater snails were tested for trematode stages and for Ehrlichia risticii DNA using a nested PCR assay. Snails were collected monthly from two Potomac horse fever (PHF) endemic locations in northern California (Montague and Weed). The trematode infection rate varied between 40 and 93.3% in large snails (shell size >15mm) and between 0 and 13.3% in small snails (<15mm). The highest trematode infection rate for large and small snails was recorded in September and the lowest infection rate for large snails was recorded in June (Weed) and October (Montague). The E. risticii PCR infection rate among small snails from both sites was similar and varied monthly between 0 and 3.3%. The PCR infection rate for large snails from Weed was high in May (20.0%) and decreased progressively until November (10.0%). The PCR infection rate for large snails from Montague was 5.0% in May, 26.3% in August and 16. 7% in October. PCR-positive snails were always related to the microscopic detection of trematode stages (virgulate cercariae). This study provides evidence that J. yrekaensis are infected with trematode cercariae that harbor E. risticii. The number of snails harboring trematode stages and the number of PCR positive snails varied with the size of the snails, the month of collection, and the geographic origin.

Ehrlichia chaffeensis expresses an immunoreactive protein homologous to the Escherichia coli GroEL protein.

A clone expressing a 58-kDa protein reactive with human convalescent-phase serum was isolated from a recombinant library of Ehrlichia chaffeensis, the etiologic agent of human ehrlichiosis. Sequencing identified two open reading frames, one encoding a 10.3-kDa polypeptide consisting of 94 amino acids and another encoding a 58-kDa polypeptide consisting of 550 amino acids. The sequences of the 10.3- and 58-kDa polypeptides were homologous to those of the Escherichia coli GroES and GroEL heat shock proteins, respectively.

Field evidence for density-dependent facilitation amongst Ixodes ricinus ticks feeding on sheep.

Engorged Ixodes ricinus nymphs collected from sheep resident in an upland UK field site were significantly lighter than nymphs that engorged on previously tick-naïve sheep, indicating that site-resident sheep continually exposed to ticks acquired anti-tick resistance. The weights of engorged nymphs that fed on naturally tick-resistant sheep increased significantly, however, when increasingly high numbers of adult female ticks fed on the sheep during seasonal peaks of tick activity. This relationship was unaffected by variations in nymph weight amongst individual sheep, between seasons and years, and potential effects of sheep infection with Ehrlichia phagocytophila; this suggests that high adult tick infestations may directly inhibit the expression of acquired anti-tick resistance by sheep. The length, width and weight of adult ticks and the scutum length of adult females were linearly related to their weight as an engorged nymph. The mean scutum length of adult female ticks feeding on sheep in the field site was greater than that of adult females obtained from engorged nymphs collected from sheep of the same site. This suggests that larger ticks have a survival advantage and that I. ricinus ticks exhibit density-dependent intraspecific facilitation at high infestation levels with potential consequences for the transmission of tick-borne diseases.

Natural Ehrlichia phagocytophila transmission coefficients from sheep 'carriers' to Ixodes ricinus ticks vary with the numbers of feeding ticks.

In a longitudinal study in a UK upland site, 38% of adult sheep were detected as infected with the tick-borne bacterium Ehrlichia phagocytophila by PCR of blood samples. Infection prevalence declined significantly with sheep age but varied significantly and non-linearly with the number of adult Ixodes ricinus ticks feeding per sheep. These findings suggested that under conditions of natural repeated tick-borne challenge sheep remain partially susceptible to re-infections, but the likelihood of re-infection depended on the numbers of feeding ticks. Transmission efficiency from sheep to immature ticks also varied significantly and non-linearly with the number of adult ticks feeding per sheep: transmission efficiency was almost zero in sheep with low adult tick infestations rising to 30% at certain levels of adult tick infestation. Infection intensity in infected engorged immature ticks also varied with the number of adult ticks feeding per sheep, but neither prevalence nor intensity of infection in engorged ticks were related to sheep blood PCR result. These findings suggest that variation in the numbers of ticks feeding per sheep may influence E. phagocytophila transmission by direct effects on transmission at the tick-host interface.

Detección molecular de patógenos emergentes de importancia médica y veterinaria en garrapatas capturadas sobre caballos domésticos / Molecular detection of emerging pathogens of medical and veterinary importance in ticks found in domestic horses

En la actualidad son varias las especies de patógenos emergentes de importancia médica y veterinaria transmitidos por garrapatas. Los estudios sobre estos agentes y sus enfermedades han sido escasos en Cuba. Conocer la presencia de algunos de estos patógenos en garrapatas cubanas que afectan el ganado equino. Se procesaron 95 garrapatas colectadas de caballos domésticos, conservadas en alcohol e identificadas taxonómicamente según claves convencionales. A cada una se le realizó extracción de ADN y posteriormente diferentes reacciones en cadena de la polimerasa utilizando cebadores específicos para los grupos microbianos Borrelia burgdorferi sensu lato, Anaplasma-Ehrlichia, y Babesia-Theileria. Cada uno de los productos de las reacciones en cadena de la polimerasa fue sometido a hibridaciones en línea reversa utilizando sondas para cada grupo en cuestión, así como específicas para las principales especies de estos. Las garrapatas estudiadas pertenecían a las especies Dermacentor (Anocentor) nitens (60 por ciento), Amblyomma cajennense (38 por ciento) y Rhipicephalus (Boophilus) microplus (2 por ciento). Se detectaron 7 garrapatas Dermacentor (Anocentor) nitens infectadas con bacterias del grupo Anaplasma/Ehrlichia, y no se pudo identificar la especie en cuestión con las sondas utilizadas. Una de estas garrapatas estaba además coinfectada con Babesia bovis. Se sugiere la circulación de una nueva especie de Anaplasma o Ehrlichia no reportada antes en Cuba, por lo que se necesita estudiar un número mayor de garrapatas, así como la incorporación de nuevas sondas en la hibridación en línea reversa u otras metodologías que permitan conocer con exactitud las especies que pudiesen afectar hoy día los caballos domésticos.

At present, there are several tick-borne emerging pathogen species of medical and veterinary importance. Few studies on these agents and its diseases have been made in Cuba. To determine the presence of some of these pathogens in Cuban ticks existing in the equine cattle. Ninety five ticks collected from domestic use horses were processed, preserved in alcohol and taxonomically identified according to the set classifications. Their DNA was extracted and subjected to several polymerase chain reactions with specific primers for microbial groups Borrelia burgdorferi sensu lato, Anaplasma-Ehrlichia, y Babesia-Theileria. Each of the products from polymerase chain reactions underwent reverse line blot hybridation using probes for each group as well as specific probes for the main species included in these groups. The studied ticks belonged to Dermacentor (Anocentor) nitens (60 percent), Amblyomma cajennense (38 percent) y Rhipicephalus (Boophilus) microplus (2 percent). Seven Dermacentor (Anocentor) nitens ticks infested with Anaplasma/Ehrlichia bacteria were detected but the species in question could not be detected by the used probes. One of these ticks was also co-infested with Babesia bovis. It is suggested that a new species of Anaplasma o Ehrlichia, not reported in Cuba before now, is circulating, so studying a higher number of ticks is needed and new probes in reverse line blot hybridation or other methodologies must be incorporated to allow exactly determining the species that may affect the Cuban domestic horses at present.