Ehrlichia ewingii sp. nov., the etiologic agent of canine granulocytic ehrlichiosis.

The 16S rRNA gene was amplified, cloned, and sequenced from the blood of two dogs that were experimentally infected with the etiologic agent of canine granulocytic ehrlichiosis. The 16S rRNA sequence was found to be unique when it was compared with the sequences of other members of the genus Ehrlichia. The most closely related species were Ehrlichia canis (98.0% related) and the human ehrlichiosis agent (Ehrlichia chaffeensis) (98.1% related); all other species in the genus were found to be phylogenetically much more distant. Our results, coupled with previous serologic data, provide conclusive evidence that the canine granulocytic ehrlichiosis agent is a new species of the genus Ehrlichia that is related to, but is distinct from, E. canis and all other members of the genus. We propose the name Ehrlichia ewingii sp. nov.; the Stillwater strain is the type strain.     

Molecular evidence of coinfection of Borrelia burgdorferi sensu lato,human granulocytic ehrlichiosis agent,and Babesia microti in ticks from northwestern Poland

To assess the potential risk for tick-borne agents, Ixodes ricinus were collected from 2 sites in northwestern Poland. The ticks were tested by polymerase chain reaction for coinfection with Borrelia burgdorferi sensu lato (s. l.), human granulocytic ehrlichiosis (HGE) agent, and Babesia microti. Of the 533 processed ticks, 16.7% were positive for B. burgdorferi s. l., 13.3% for B. microti, and 4.5% for the HGE agent. Twenty ticks were coinfected with 2 or 3 of the pathogens.     

Cutting edge: infection by the agent of human granulocytic ehrlichiosis prevents the respiratory burst by down-regulating gp91phox

The agent of human granulocytic ehrlichiosis (HGE) is an emerging tick-borne pathogen that resides in neutrophils and can be cultured in a promyelocytic (HL-60) cell line. In response to microbes, polymorphonuclear leukocytes normally activate the NADPH oxidase enzyme complex and generate superoxide anion (O2-). However, HL-60 cells infected with HGE bacteria did not produce O2- upon activation with PMA. RT-PCR demonstrated that HGE organisms inhibited mRNA expression of a single component of NADPH oxidase, gp91phox, and FACS analysis showed that plasma membrane-associated gp91phox protein was reduced on the infected cells. Infection with HGE organisms also decreased gp91phox mRNA levels in splenic neutrophils in a murine model of HGE, demonstrating this phenomenon in vivo. Therefore, HGE bacteria repress the respiratory burst by down-regulating gp91phox, the first direct inhibition of NADPH oxidase by a pathogen.     

The mouse as a model for investigation of human granulocytic ehrlichiosis: current knowledge and future directions

The use of laboratory mice to investigate correlates of infectious disease, including infection kinetics, cellular alterations, cytokine profiles, and immune response in the context of an intact host has expanded exponentially in the last decade. A marked increase in the availability of transgenic mice and research tools developed specifically for the mouse parallels and enhances this research. Human granulocytic ehrlichiosis (HGE) is an emerging, zoonotic disease caused by tick-borne bacteria. The HGE agent (Anaplasma phagocytophila) is one of two recognized pathogens to cause human granulocytic ehrlichiosis (HGE). The mouse model of HGE complements in vitro tissue culture studies, limited in vivo large animal studies, and ex vivo studies of human and ruminant neutrophils, and promises new avenues to approach mechanisms of disease. In the overview reported here, we focus principally on current research into HGE pathogenesis using the mouse model. Included is a discussion of current changes in ehrlichial classification and nomenclature, a review of ehrlichial biology and ecology, and highlights of clinical disease in animals and people.     

Detection of natural foci of babesiosis and granulocytic ehrlichiosis in Russia

The use of microscopy, infection of golden hamsters and the polymerase chain reaction made it possible to find out that about 30% of common red-backed voles (Clethrionomys glareolus), inhabiting the taiga forests of the southern part of the Western Urals (the Chusovskoi district of the Perm region), were infected with Babesia microti and simultaneously (a third of them) with Ehrlichia (Cytoecetes) phagocytophila, the causative agent of granulocytic ehrlichiosis. The sequencing of 18S rDNA of strain "Mys", isolated in Russia, revealed its identity to American B. microti strain GI, pathogenic for humans. The main vector supporting the circulation of B. microti in the natural foci in the region where these investigations were conducted was, seemingly, the tick Ixodes trianguliceps, Thus, for the first time the data proving the presence of reservoir hosts infected with B. microti and granulocytic E. phagocytophila, pathogenic for humans, in Russia were presented.     

Search for Borrelia sp. in ticks collected from potential reservoirs in an urban forest reserve in the State of Mato Grosso do Sul,Brazil: a short report

A total of 128 ticks of the genus Amblyomma were recovered from 5 marsupials (Didelphis albiventris) - with 4 recaptures - and 17 rodents (16 Bolomys lasiurus and 1 Rattus norvegicus) captured in an urban forest reserve in Campo Grande, State of Mato Grosso do Sul, Brazil. Of the ticks collected, 95 (78.9%) were in larval form and 22 (21.1%) were nymphs; the only adult (0.8%) was identified as A. cajennense. Viewed under dark-field microscopy in the fourth month after seeding, 9 cultures prepared from spleens and livers of the rodents, blood of the marsupials, and macerates of Amblyomma sp. nymphs revealed spiral-shaped, spirochete-like structures resembling those of Borrelia sp. Some of them showed little motility, while others were non-motile. No such structures could be found either in positive Giemsa-stained culture smears or under electron microscopy. No PCR amplification of DNA from those cultures could be obtained by employing Leptospira sp., B. burgdorferi, and Borrelia sp. primers. These aspects suggest that the spirochete-like structures found in this study do not fit into the genera Borrelia or Leptospira, requiring instead to be isolated for proper identification.     

Causative agent of human diphyllobothriasis--Diphyllobothrium klebanovskii sp. n

A new cestode species, Diphyllobothrium klebanovskii sp. n., is described on the basis of experimental examination and morphological study of 51 specimens expelled from men in lower part of the Amur river and 18 specimens expelled from golden hamsters. The larval stage of this cestode is plerocercoid of type F, which is localized in muscles of Oncorhynchus keta and O. gorbuscha. D. klebanovskii sp. n. is a marine species, it differs from D. latum and other species of the genus Diphyllobothrium.     

Infections of granulocytic ehrlichiae and Borrelia burgdorferi in white-tailed deer in Connecticut

Serum or whole blood samples, obtained from 141 white-tailed deer (Odocoileus virginianus) in Connecticut (USA) during 1980, 1991, and 1996, were analyzed to detect past or current infections of Ehrlichia phagocytophila genogroup organisms and Borrelia burgdorferi. When the BDS or NCH-1 strains of granulocytic ehrlichiae were used separately in indirect fluorescent antibody (IFA) staining methods, antibody positivity rates varied from 25 to 64% in 1991 and 1996, respectively. All 50 sera tested from 1980 collections were negative. Although percentages of sera with B. burgdorferi antibodies, as detected by an enzyme-linked immunosorbent assay, also differed (23 to 53%), there were coexisting antibodies to both bacteria in 20 (49%) of 41 sera. In tests on specificity, 19 deer sera with ehrlichial antibodies also were tested by IFA staining procedures for Anaplasma marginale antibodies; one serum with a titer of 1:5,120 to ehrlichial antigen reacted to A. marginale antigen at a serum dilution of 1:320. In parallel analyses of 69 sera, results of Western blot analyses for ehrlichial infections in deer were concordant (72% agreement) with those of IFA staining methods containing ehrlichial antigen. All positive immunoblots showed bands to peptides of the NCH-1 strain of the human granulocytic ehrlichiosis (HGE) agent having molecular masses of about 44, 105, or 110 kDa. In polymerase chain reaction (PCR) studies of blood samples from 63 deer, 11 (18%) specimens were positive for 16S ribosomal DNA of an Ehrlichia phagocytophila genogroup organism, whereas 23 (37%) samples were positive for the DNA of the 44 kDa gene of the HGE agent. White-tailed deer are exposed to different tick-borne bacteria in areas where Ixodes scapularis ticks are abundant and may, in some instances, have had concurrent infections.     

Changes in expression of the 44-kilodalton outer surface membrane antigen (p44 kD) for monitoring progression of infection and antimicrobial susceptibility of the human granulocytic ehrlichiosis (HGE) agent in HL-60 cells

Changes in human granulocytic ehrlichiosis (HGE)-specific major outer membrane protein (p44 kD) were assayed by Western blot analysis in HL-60 cells in vitro infected by the HGE agent. Time course study demonstrated that the expression of p44 preceded the rise in cell infection as determined by the presence of intracellular morulae. To test whether the expression of p44 may be suitable for evaluating the effects of antibiotics in vitro, three recent isolates of the HGE agent were exposed to doxycycline and ampicillin during culture with HL-60 cells. Loss of infection concurrent with disappearance of the 44 kD protein was found with doxycycline treatment. In contrast, ampicillin treatment had no discernible effects on infection or 44 kD expression. There was excellent agreement between infection, as measured by morulae, and 44 kD expression (coefficient of correlation r = 0.97, p < 0.01). Following treatment with doxycycline, the 44 kD protein disappeared with an estimated t1/2 of approximately 24-30 h, which was considerably shorter than a t1/2 of >60 h calculated for loss of morulae. Measurement of p44 expression may be a more rapid and simple assay to determine antibiotic susceptibility of the HGE agent in cell culture. Furthermore, it may be used to indicate the presence of infection before morulae are apparent.     

Pre-release evaluation of Heikertingerella sp. as a potential biocontrol agent for Tecoma stans in South Africa

Native to Central America, Tecoma stans (L.) Juss ex Kunth var. stans (Bignoniaceae) is a small tree that is invasive in South Africa and neighbouring countries. The plant was targeted for biological control in South Africa in 2003, with two insect agents released and established so far. The root-feeding flea beetle, Heikertingerella sp. (Coleoptera: Galerucinae: Alticini), was imported from Mexico as an additional biocontrol agent and its biology and host specificity was assessed under quarantine conditions. The beetle displayed a generation time (i.e. from adult to adult) of 49 to 67 days, ensuring four annual generations under laboratory conditions. The beetle's larval and adult stages inflicted high levels of damage on the root system and the leaves of T. stans, respectively. No-choice tests with 40 test-plant species revealed adult feeding on only two non-target species, Tecoma × alata and T. capensis (Thunb.) Spach, with feeding four times higher on T. stans. Larvae developed to adulthood on T. stans only. Multi-choice tests involving the three Tecoma species confirmed these trends, demonstrating that Heikertingerella sp. is host specific. Since T. × alata is a hybrid of T. stans with invasive tendencies, any unlikely attacks by Heikertingerella sp. would be inconsequential in South Africa. The native T. capensis, which suffered little leaf damage and produced no F1 adults, is also at minimal risk of attack. We conclude that Heikertingerella sp. is a suitable biocontrol agent for T. stans and that permission for its release in South Africa be sought.     

The flagellin gene of Borrelia miyamotoi sp. nov. and its phylogenetic relationship among Borrelia species

Abstract We have cloned and sequenced the flagellin gene from Borrelia miyamotoi strain HT31 and compared it with previously published flagellin sequences. Sequence similarity analysis demonstrated that strain HT31 is phylogenetically distant from the three species of Lyme disease borreliae and is deeply branched into the relapsing fever borrelia cluster. The result was in full agreement with the classification of Borrelia strains using 16S rRNA sequences. This finding indicates that a phylogenetic analysis using flagellin gene sequences might be useful for classification of Borrelia strains.     

Borrelia sp. in ticks recovered from white-tailed deer in Alabama.

Six hundred sixty-five hunter-killed white-tailed deer (Odocoileus virginianus) from 18 counties in Alabama (USA) were examined for ticks. Most of the collections were made at state-operated wildlife management areas. Four species of ticks (n = 4,527) were recovered: the lone star tick Amblyomma americanum (n = 482); the Gulf Coast tick A. maculatum (n = 11); the winter tick Dermacentor albipictus (n = 1,242); and the black-legged tick Ixodes scapularis (n = 2,792). Fifty-six percent of the ticks (n = 2,555) were examined for Borrelia sp. spirochetes using an immunofluorescent, polyclonal antibody assay. Spirochetes were detected in I. scapularis (five females, seven males) from Barbour, Butler, Coosa, and Lee counties and A. americanum (four males, four nymphs) from Hale, Lee, and Wilcox counties. Area-specific prevalences in ticks were as high as 3.3% for I. scapularis and 3.8% for A. americanum.     

Genomic-assisted characterisation of Pseudomonas sp. strain Pf4, a potential biocontrol agent in hydroponics

In an attempt to select potential biocontrol agents against Pythium spp. and Rhizoctonia spp. root pathogens for use in soilless systems, 12 promising bacteria were selected for further investigations. Sequence analysis of the 16S rRNA gene revealed that three strains belonged to the genus Enterobacter, whereas nine strains belonged to the genus Pseudomonas. In in vitro assays, one strain of Pseudomonas sp., Pf4, closely related to Pseudomonas protegens (formerly Pseudomonas fluorescens), showed noteworthy antagonistic activity against two strains of Pythium aphanidermatum and two strains of Rhizoctonia solani AG 1-IB, with average inhibition of mycelial growth >80%. Strain Pf4 was used for in vivo treatments on lamb’s lettuce against R. solani root rot in small-scale hydroponics. Pf4-treated and untreated plants were daily monitored for symptom development and after two weeks of infection, a significant protective effect of Pf4 against root rot was recorded. The survival and population density of Pf4 on roots were also checked, demonstrating a density above the threshold value of 10⁵?CFU?g^?1 of root required for disease suppression. Known loci for the synthesis of antifungal metabolites, detected using PCR, and draft-genome sequencing of Pf4 demonstrated that Pseudomonas sp. Pf4 has the potential to produce an arsenal of secondary metabolites (plt, phl, ofa and fit-rzx gene clusters) very similar to that of the well-known biocontrol P. protegens strain Pf-5.     

Avian reservoirs and zoonotic potential of the emerging human pathogen Helicobacter canadensis

A polyphasic identification approach was used to investigate the taxonomic position of Campylobacter-like isolates recovered from barnacle geese (Branta leucopsis) and Canada geese (Branta candensis). Seven strains were selected from a collection of 21 isolates and analyzed by extensive phenotypic testing; four strains were characterized by 16S rRNA gene sequence analysis. The results clearly identified the bird isolates as Helicobacter canadensis, recently described as an emerging human pathogen. This is the first report of an animal reservoir for this organism and of its presence in Europe and confirms the zoonotic potential of H. canadensis.     

Relationships of a Novel Lyme Disease Spirochete, Borrelia spielmani sp. nov., with Its Hosts in Central Europe

To determine whether the pathogenic variant of Lyme disease spirochetes, isolate A14S, is perpetuated in a particular reservoir-vector relationship, we screened vector ticks in various Central European sites for a related spirochete and determined its host association. A14S-like spirochetes infect numerous questing ticks in the Petite Camargue Alsacienne (PC). They frequently infect dormice, but no mice or voles. Garden dormice appear to be better reservoir hosts for A14S-like spirochetes than for Borrelia afzelii, because these spirochetes are retained longer and infect ticks more readily. Spirochetes associated with garden dormice in the PC site form a homologous entity with those isolated from a human patient in The Netherlands. Its unique biological relationship together with previous genetic characterization justifies designating this dormouse-associated genospecies as a distinct entity. Garden dormice serve as the main reservoir hosts of a novel genospecies, Borrelia spielmani sp. nov., one of several that cause Lyme disease in people.     

Trichilogaster sp. [Hymenoptera: Pteromalidae], a potential biocontrol agent for the weedAcacia pycnantha [Fabaceae]

The gall wasp,Trichilogaster sp., was imported from Australia to assess its potential as an agent for the control of the invasive shrub/treeAcacia pycnantha Benth. in South Africa. Host specificity tests indicate safety for release; of 19 tree/shrub species tested, including 16 species closely related toA. pycnantha, galls developed only onA. pycnantha. However, galling intensity remained consistently low on the host plant; only 21–29% of the branches exposed to the wasp were galled during 3 years of rearing. Neither the prolonged presence of males in test cages (someTrichilogaster species are thelytokous) nor the stage of maturity of reproductive buds exposed to oviposition affected the percentage of branches galled. It is not recommended thatTrichilogaster sp. be released before the possibility of insect-plant homeostasis or mis-matching of wasp and host plant populations/strains/subspecies is investigated, especially since galling intensities of 30% were ineffective in reducing seed production of a relatedTrichilogaster species/Acacia association.      

Membrane potential in human myeloid leukemia cell line ML-1: responsiveness of granulocytic and monocytic differentiated cells.

The membrane potential responsiveness of human myeloid leukemia cells (ML-1 line) was studied with the voltage sensitive fluorescent dye diS-C3-(5). The experimental procedure used in this study enabled us to assess the magnitude of the membrane potential change in cells treated with ouabain, 12-0-tetradecanoylphorbol-13-acetate (TPA) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), relative to the membrane potential in the untreated control. Inhibition of the Na, K-ATPase by ouabain was followed by a (20 +/- 4) mV depolarization. In undifferentiated homogeneous cell population TPA caused a (19.4 +/- 4.4) mV depolarization while FMLP had virtually no effect. Cells in which granulocytic or monocytic differentiation was induced by retinoic acid or 1,25-dihydroxyvitamin D3 exhibited under the effect of TPA a (57.8 +/- 7.1) mV and (34.8 +/- 10.9) mV depolarization, respectively. A very small transient depolarization was also observed up on treating of the cells with FMLP. The changes in the membrane potential responsiveness in the induced cells are obviously connected with the cell differentiation.