Novel Ehrlichia sp. detected in Magellanic penguins (Sphenicus magellanicus) and in the seabird tick Ixodes uriae from Magdalena Island, southern Chile.

Ehrlichia spp. are obligatory intracellular microorganisms that infect hematopoietic, endothelial or blood cells of mammals. Ticks are the only vectors of these agents in nature. To date, the role of birds and their associated ticks as reservoirs of ehrlichiae remains almost unexplored. In this study, we performed a molecular screening for bacteria of Anaplasmataceae family in samples of spleen (n = 72) and lung (n = 17), recovered from 72 carcasses of Magellanic penguins (Spheniscus magellanicus) in Brazil and Chile. One apparently unengorged tick (Ixodes uriae) was also collected while wandering upon one of the carcasses and submitted to molecular analyses as well. Through conventional and nested PCR protocols three genes (16S rRNA, dsb and groEL) of a new Ehrlichia sp. were partially characterized upon organs of three penguins and in the tick coming from Magdalena Island (Chile). First matches after BLASTn comparisons showed that our sequences share 99.4% (16S rRNA), 94.6% (groEL) and 79.3% (dsb) of identity with "Candidatus Ehrlichia ornithorhynchi", Ehrlichia sp. NS101 and Ehrlichia canis CCZ, respectively. Matrixes of genetic distance including other representatives of the Ehrlichia genus point a 99.4%, 94.0%, and 80.0% of identity with 16S rRNA, groEL and dsb genes from Ehrlichia sp. It25, Ehrlichia sp. NS101, and Ehrlichia chaffeensis San Louis, respectively. A Bayesian phylogenetic analysis of Anaplasmataceae 16S rRNA gene places the detected Ehrlichia sp. into a group with Ehrlichia sp. BAT and Ehrlichia sp. Natal. Although depicting different topologies, Bayesian unrooted phylogenetic trees constructed for groEL and dsb genes position this Ehrlichia sp. into well-supported branches, which reinforces the finding of a new taxon. For the moment, any pathogenic effect of this new Ehrlichia sp. on penguins is still unknown. However, this fact becomes important to assess from a conservation point of view since populations of Magellanic penguins are currently threatened and in an ongoing decrease.

SEROLOGICAL SURVEY OF Ehrlichia SPECIES IN DOGS, HORSES AND HUMANS: ZOONOTIC SCENERY IN A RURAL SETTLEMENT FROM SOUTHERN BRAZIL / Investigação sorológica de espécies de Ehrlichia em cães, equinos e humanos de um assentamento rural do sul do Brasil

SUMMARY The aims of this study were to determine the seroprevalence of Ehrlichia spp. and risk factors for exposure in a restricted population of dogs, horses, and humans highly exposed to tick bites in a Brazilian rural settlement using a commercial ELISA rapid test and two indirect immunofluorescent assays (IFA) with E. canis and E. chaffeensis crude antigens. Serum samples from 132 dogs, 16 horses and 100 humans were used. Fifty-six out of 132 (42.4%) dogs were seropositive for E. canis. Dogs > one year were more likely to be seropositive for E. canis than dogs ≤ one year (p = 0.0051). Ten/16 (62.5%) and 8/16 (50%) horses were seropositive by the commercial ELISA and IFA, respectively. Five out of 100 (5%) humans were seropositive for E. canis and E. chaffeensis. Rhipicephalus sanguineus (n = 291, 97.98%) on dogs and Amblyomma cajennense (n = 25, 96.15%) on horses were the most common ticks found. In conclusion, anti-Ehrlichia spp. antibodies were found in horses; however, the lack of a molecular characterization precludes any conclusion regarding the agent involved. Additionally, the higher seroprevalence of E. canis in dogs and the evidence of anti-Ehrlichia spp. antibodies in humans suggest that human cases of ehrlichiosis in Brazil might be caused by E. canis, or other closely related species. .

RESUMO Objetivou-se determinar a soroprevalência de Ehrlichia spp. e os fatores de risco associados a exposição em uma população restrita de cães, cavalos e humanos altamente expostos a picadas de carrapatos em um assentamento rural brasileiro utilizando um teste comercial de ELISA rápido e dois testes de imunofluorescência indireta (IFI) com antígenos brutos de E. canis e E. chaffeensis. Amostras de soro de 132 cães, 16 cavalos e 100 humanos foram utilizadas. Cinquenta e seis/132 (42,4%) cães foram soropositivos para E. canis. Cães > um ano apresentaram mais chance de serem soropositivos para E. canis do que cães ≤ um ano (p = 0,0051). Dez/16 (62,5%) e 8/16 (50%) cavalos foram soropositivos pelo ELISA comercial e IFI, respectivamente. Cinco/100 (5%) humanos foram soropositivos para E. canis e E. chaffeensis. Rhipicephalus sanguineus (n = 291, 97,98%) nos cães e A. cajennense (n = 25, 96,15%) nos cavalos foram os carrapatos mais encontrados. Concluindo, anticorpos anti-Ehrlichia spp. foram encontrados em cavalos; entretanto, a ausência de uma caracterização molecular impede qualquer conclusão sobre agente envolvido. Além disso, a alta soroprevalência de E. canis em cães e a evidência de anticorpos anti-Ehrlichia sp. em humanos, sugere que os casos de erliquiose humana no Brasil possam ser causados por E. canis ou outra espécie intimamente relacionada. .

Polyphasic taxonomy.

Several organisms from a number of prokaryotic and eukaryotic groups have presented problems for systematists for a long time. Both phenotypic and genotypic methods for sorting out these relationships have been employed. There are limitations with each method when taken alone. Since the purpose of systematics is to determine the correct genealogical relationships among biological organisms, it is necessary to use all available means to arrive at consensus associations, and polyphasic taxonomy, which takes into consideration both methods, is a rational approach. In this short article, we provide a number of examples where polyphasic taxonomy is serving as the means of arriving at the desired consensus.

Ehrlichial proliferation and acute hepatocellular necrosis in immunocompetent mice experimentally infected with the HF strain of Ehrlichia, closely related to Ehrlichia chaffeensis.

Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis, is closely related to the HF strain of Ehrlichia isolated from ticks in Japan. In this study, BALB/c mice inoculated intraperitoneally with the HF strain developed severe illness and died at about day 9 post-inoculation. At necropsy, diffuse liver necrosis was evident. Ehrlichial microcolonies were observed in endothelial cells, monocytes and macrophages of the liver, bone marrow, spleen, thymus, and large and small intestine. Immunocompetent mice infected with the HF strain would provide a useful model for studying pathogenesis and immunity in acute and severe ehrlichiosis caused by E. chaffeensis and related Ehrlichia spp.

New Ehrlichia species closely related to Ehrlichia chaffeensis isolated from Ixodes ovatus ticks in Japan.

Seven Ehrlichia strains (six HF strains and one Anan strain) that were obtained from laboratory mice by intraperitoneally inoculating homogenates of adult Ixodes ovatus collected in Japan were characterized. 16S rRNA sequences of all six HF strains were identical, and the sequences were 99.7, 98.2, and 97.7% identical to those of Anan strain, Ehrlichia chaffeensis (human monocytic ehrlichiosis agent), and E. muris, respectively. Partial GroEL amino acid sequencing also revealed that the six HF strains had identical sequences, which were 99.0, 98.5, and 97.3% identical to those of E. chaffeensis, the Anan strain, and E. canis, respectively. All HF strains were lethal to mice at higher dosages and intraperitoneal inoculation, whereas the Anan or E. muris strain induced only mild clinical signs. Light and electron microscopy of moribund mice inoculated with one of the HF strains revealed severe liver necrosis and the presence of numerous ehrlichial inclusions (morulae) in various organs. The study revealed that members of E. canis genogroup are naturally present in Ixodes ticks. HF strains that can cause severe illness in immunocompetent laboratory mice would be valuable in studying the pathogenesis and the roles of both cellular and humoral immune responses in ehrlichiosis caused by E. canis genogroup.

Reactivity of human sera to different strains of granulocytic ehrlichiae in immunodiagnostic assays.

Sera from 35 patients diagnosed with human granulocytic ehrlichiosis in Connecticut were tested by indirect IFA staining methods with 5 strains of Ehrlichia equi or the human granulocytic ehrlichiosis agent to assess the suitability of different strains in laboratory analyses. Antigens included horse-derived infected neutrophils (MRK and BDS strains) and human isolates cultured in human promyelocytic leukemia cells (NCH-1, RCH, and Webster). Of 35 sera, 23 (65.7%) reacted to all 5 strains. Seropositivity was highest (97.1%) in assays that contained the MRK strain from California and lowest (71. 4%) in tests with the NCH-1 strain from Nantucket, Massachusetts. In parallel testing of 32 sera with the NCH-1 strain by indirect IFA and Western blot analyses, results were concordant for 30 samples (93.8%). All strains of ehrlichiae can be used in IFA analyses for antibody detection, but assay sensitivity varied with the strain used.

Ultrastructural differentiation of the genogroups in the genus Ehrlichia.

Ultrastructural characteristics of 15 strains and isolates of ehrlichiae belonging to three genogroups, or clades of genetically related organisms united in the genera Ehrlichia, Cowdria, Anaplasma, Neorickettsia and a strain of Wolbachia pipientis which represents a fourth genogroup in this cluster of species, were studied in continuous cell culture or in vivo: E. canis (Oklahoma strain and VHE isolate), E. muris (AS 145), E. chaffeensis (Arkansas, 91HE17 and Sapulpa), human granulocytic ehrlichiae (HGE)(BDS, 96HE27, 96HE37, #54, #55 and #72), E. equi (MRK), E. sennetsu (Miyayama), E. risticii (HRC-IL). Wolbachia pipientis was studied in the naturally infected Aedes albopictus mosquito cell line Aa23. All organisms were similar in the normal ultrastructure of individual cells and in the ability to form abnormal, pathological ehrlichial cells of the same type irrespective of the species. Normally all ehrlichiae studied in cell culture existed in two morphological forms - reticulate and dense-cored cells, both of which could divide by binary fission. Most alterations were related to their membranes, especially the cell wall. Differences in the structure of intravacuolar microcolonies (morulae) of ehrlichiae and their inter-relations with the host cells allowed differentiation of the genogroups: the E. canis-E. chaffeensis-E. muris genogroup formed large morulae, with many ehrlichiae, often suspended in a fibrillar matrix, and the host cell mitochondria and endoplasmic reticulum usually aggregated near the morulae and were in contact with the morula membrane; the E. phagocytophila-E. equi-HGE group morulae had no fibrillar matrix, no contacts with host cell mitochodria, and they did not aggregate around the morulae; E. sennetsu-E. risticii group usually developed in small individual vacuoles that did not fuse with each other and divided along with the ehrlichiae.

Emerging pathogens associated with tick-borne infections

In recent years, several microbial agents have been identified that result in significant morbidity and mortality. The newly recongnized tick borne infections, babesiosis and ehrlichiosis, may be transmitted by the same tick that transmits Borrelia burgdorferi and simultaneous infections may occur. Babesia are intraerythrocytic protozoa that may cause severe hemolytic anemia, whereas Ehrlichia, depending on the species, may infect either monocytes or granulocytes, with associated leukopenia, thrombocytopenia and anemia. Improved laboratory surveillance is urgently needed to assess the prevalence of these worldwide pathogens in order to institute appropriate infection control efforts.

Direct cultivation of the causative agent of human granulocytic ehrlichiosis.

BACKGROUND: Human granulocytic ehrlichiosis is a potentially fatal tick-borne infection that has recently been described. This acute febrile illness is characterized by myalgias, headache, thrombocytopenia, and elevated serum aminotransferase levels. The disease is difficult to diagnose because the symptoms are non-specific, intraleukocytic inclusions (morulae) may not be seen, and the serologic results are often initially negative. Little is known about the causative agent because it has never been cultivated. METHODS: We studied three patients with symptoms and laboratory findings suggestive of human granulocytic ehrlichiosis, including unexplained fever after probable exposure to ticks, granulocytopenia, and thrombocytopenia. Peripheral blood was examined for ehrlichia microscopically and with use of the polymerase chain reaction (PCR). Blood was inoculated into cultures of HL60 cells (a line of human promyelocytic leukemia cells), and the cultures were monitored for infection by Giemsa staining and PCR. RESULTS: Blood from the three patients, only one of whom had inclusions suggestive of ehrlichia in neutrophils, was positive for human granulocytic ehrlichiosis on PCR. Blood from all three patients was inoculated into HL60 cell cultures and caused infection, with intracellular organisms visualized as early as 5 days after inoculation and cell lysis occurring within 12 to 14 days. The identity of the cultured organisms was confirmed by immunofluorescence microscopy, PCR analysis, and DNA sequencing. DNA from the infected cells was sequenced in regions of the 16S ribosomal gene reported to differ between the agent of human granulocytic ehrlichiosis and closely related species, including Ehrlichia equi and E. phagocytophila which cause infection in animals. The sequences from all three human isolates were identical and differed from the strain of E. equi studied in having guanine rather than adenine at nucleotide 84. CONCLUSIONS: We describe the cultivation of the agent of human granulocytic ehrlichiosis in cell culture. The ability to isolate this organism should lead to a better understanding of the biology, treatment, and epidemiology of this emerging infection.

Western immunoblot analysis of Ehrlichia chaffeensis, E. canis, or E. ewingii infections in dogs and humans.

Ehrlichia chaffeensis, E. canis, and E. ewingii are genetically closely related, as determined by 16S rRNA gene base sequence comparison, but they exhibit biologic differences. E. chaffeensis is the etiologic agent of human ehrlichiosis. E. canis and E. ewingii cause two distinctly different forms of canine ehrlichiosis and infect different types of leukocytes, monocytes and granulocytes, respectively. E. chaffeensis can also infect dogs. In the study, Western immunoblot analysis of sera from dogs inoculated with E. chaffeensis, E. canis, or E. ewingii was performed to determine antigenic specificity and the intensities of the reactions to purified E. chaffeensis and E. canis antigens. At 2 to 3 weeks postexposure, antisera from four dogs inoculated with E. chaffeensis reacted with 64-, 47-, 31-, and 29-kDa proteins of E. chaffeensis but reacted poorly with E. canis antigen. In contrast, at 2 to 3 weeks postexposure, antisera from four E. canis-inoculated dogs reacted strongly with the 30-kDa major antigen of E. canis but reacted poorly with proteins from E. chaffeensis. At 4 weeks postexposure, the sera from three E. ewingii-inoculated dogs showed weak binding to 64- and 47-kDa proteins of both E. chaffeensis and E. canis. Convalescent-phase sera from human ehrlichiosis patients and sera from dogs chronically infected with E. ewingii strongly reacted with similar sets of proteins of E. chaffeensis and E. canis with similar intensities. However, sera from dogs chronically infected with E. canis reacted more strongly with a greater number of E. canis proteins than with E. chaffeensis proteins. The protein specificity described in the report suggests that dogs with E. canis infections can be distinguished from E. chaffeensis-infected animals by Western immunoblot analysis with both E. canis and E. chaffeensis antigens.