Tomoko Tajima

Novel Genetic Variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a Novel Ehrlichia sp. in Wild Deer and Ticks on Two Major Islands in Japan

ABSTRACT Wild deer are one of the important natural reservoir hosts of several species of Ehrlichia and Anaplasma that cause human ehrlichiosis or anaplasmosis in the United States and Europe. The primary aim of the present study was to determine whether and what species of Ehrlichia and Anaplasma naturally infect deer in Japan. Blood samples obtained from wild deer on two major Japanese islands, Hokkaido and Honshu, were tested for the presence of Ehrlichia and Anaplasma by PCR assays and sequencing of the 16S rRNA genes, major outer membrane protein p44 genes, and groESL. DNA representing four species and two genera of Ehrlichia and Anaplasma was identified in 33 of 126 wild deer (26%). DNA sequence analysis revealed novel strains of Anaplasma phagocytophilum, a novel Ehrlichia sp., Anaplasma centrale, and Anaplasma bovis in the blood samples from deer. None of these have been found previously in deer. The new Ehrlichia sp., A. bovis, and A. centrale were also detected in Hemaphysalis longicornis ticks from Honshu Island. These results suggest that enzootic cycles of Ehrlichia and Anaplasma species distinct from those found in the United States or Europe have been established in wild deer and ticks in Japan.

Changes in concentrations of serum amyloid A protein, α1-acid glycoprotein, haptoglobin, and C-reactive protein in feline sera due to induced inflammation and surgery

To identify candidates for feline acute phase proteins, the concentrations of serum amyloid A protein (SAA), alpha 1-acid glycoprotein (alpha 1-AG), C-reactive protein (CRP), and haptoglobin (Hp) were measured in sera isolated from clinically normal and hospitalized (or diseased) cats, from cats with experimentally induced inflammation, and cats subjected to surgery for urinary diversion. Measurements were made by sandwich enzyme-linked immunosorbent assay and single radial immunodiffusion. The concentrations of SAA, alpha 1-AG, and Hp in sera from hospitalized cats were 7-11 times higher than in clinically normal cats. Similar results were obtained for the concentrations of SAA, alpha 1-AG, and Hp in cats with induced inflammation and cats subjected to surgery. By contrast, the serum concentration of feline CRP did not change significantly between clinically normal cats and hospitalized cats or inflammation-induced or post-surgery cats. Feline SAA concentration was found to increase earliest, with alpha 1-AG and Hp beginning to increase thereafter. From these results, feline SAA is concluded to be an acute phase reactant at the early stage of inflammation.

Transcript heterogeneity of the p44 multigene family in a human granulocytic ehrlichiosis agent transmitted by ticks.

ABSTRACT Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne zoonosis caused by a strain of Anaplasma phagocytophila called the HGE agent, an obligatory intracellular bacterium. The agent expresses immunodominant 44-kDa outer membrane proteins (P44s) encoded by a multigene family. The present study established an experimental process for transmission of the HGE agent from infected mice (a reservoir model) to nymphal Ixodes scapularis ticks (a biological vector) and subsequently to horses (a patient model) by the adult infected ticks. Overall, a total of 20 different p44 transcripts were detected in the mammals, ticks, and cell cultures. Among them, a transcript from a p44-18 gene was major at acute stage in mice and horses but minor in ticks. Both mRNA and protein produced from the p44-18 gene were detected in the HGE agent cultivated in HL-60 cells at 37°C, but their expression levels decreased in the organisms cultivated at 24°C, suggesting that temperature is one of the factors that influence the expression of members of the p44 multigene family. Several additional p44 transcripts that were not detected in the mammals at the acute stage of infection were detected in ticks. Phylogenetic analysis of the 20 different p44 transcripts revealed that the major transcripts found in mammals and ticks were distinct, suggesting a difference in surface properties between populations of the HGE agent in different host environments. The present study provides new information for understanding the role of the p44 multigene family in transmission of the HGE agent between mammals and ticks.

Transcriptional Analysis of p30 Major Outer Membrane Multigene Family of Ehrlichia canis in Dogs, Ticks, and Cell Culture at Different Temperatures

ABSTRACT Ehrlichia canis, an obligatory intracellular bacterium of monocytes and macrophages, causes canine monocytic ehrlichiosis. E. canisimmunodominant 30-kDa major outer membrane proteins are encoded by a polymorphic multigene family consisting of more than 20 paralogs. In the present study, we analyzed the mRNA expression of 14 paralogs in experimentally infected dogs andRhipicephalussanguineus ticks by reverse transcription-PCR using gene-specific primers followed by Southern blotting. Eleven out of 14 paralogs in E.canis were transcribed in increasing numbers and transcription levels, while the mRNA expression of the 3 remaining paralogs was not detected in blood monocytes of infected dogs during the 56-day postinoculation period. Three different groups ofR. sanguineus ticks (adult males and females and nymphs) were separately infected with E.canis by feeding on the infected dogs. In these pools of acquisition-fed ticks as well as in the transmission-fed adult ticks, the transcript from only one paralog was detected, suggesting the predominant transcription of that paralog or the suppression of the remaining paralogs in ticks. Expression of the same paralog was higher whereas expression of the remaining paralogs was lower inE. canis cultivated in dog monocyte cell line DH82 at 25°C than in E. caniscultivated at 37°C. Analysis of differential expression ofp30 multigenes in dogs, ticks, or monocyte cell cultures would help in understanding the role of these gene products in pathogenesis and E. canis transmission as well as in designing a rational vaccine candidate immunogenic against canine ehrlichiosis.

Cytokine Gene Expression by Peripheral Blood Leukocytes in Horses Experimentally Infected with Anaplasma phagocytophila

ABSTRACT Human granulocytic ehrlichiosis (HGE), a tick-borne zoonosis, is caused by an obligatory intragranulocytic bacterium, the HGE agent, a strain of Anaplasma phagocytophila. The equine model of HGE is considered valuable in understanding pathogenic and immune mechanisms of HGE. In the present study, cytokine mRNA expression by peripheral blood leukocytes (PBLs) in horses was examined during the course of infection by intravenous inoculation of A. phagocytophila or by allowing feeding by infected ticks. The p44 genes encoding the major outer membrane protein P44s of A. phagocytophila were detected by PCR in PBLs of all four horses from 4 to 20 days postexposure. During the 20-day infection period, interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) mRNA expression was upregulated in PBLs of all four horses, and IL-8 mRNA expression was upregulated in three horses. Gamma interferon, IL-10, and IL-12 p35 mRNAs were weakly expressed in only one horse each. IL-2, IL-4, IL-6, and IL-12 p40 mRNA expression , however, could not be detected in the PBLs of any of the four horses. These results suggest that IL-1β, TNF-α, and IL-8 generation during A. phagocytophila infection has a primary role in HGE pathogenesis and immunomodulation.

Molecular detection of Anaplasma phagocytophilum in cattle and Ixodes persulcatus ticks.

The tick-borne pathogen, Anaplasma phagocytophilum (A. phagocytophilum), the causative agent of human granulocytic anaplasmosis (HGA), is increasingly becoming a public health concern as an aetiological agent for emerging infectious disease. We found A. phagocytophilum infection in a pooled sample of field-collected Ixodes persulcatus (I. persulcatus) ticks from one district in Hokkaido, Japan. Thus, to further investigate the prevalence in field-collected ticks, we used PCR assays targeting the A. phagocytophilum gene encoding 44 kDa major outer membrane protein (p44) for screening of I. persulcatus ticks and samples from cattle from pastures. Out of the 281 I. persulcatus ticks, 20 (7.1%) were found to harbor A. phagocytophilum DNA. The infection rate for A. phagocytophilum in cattle was 3.4% (42/1251). In future studies, it will be necessary to investigate effects of the infection in order to understand its pathogenesis of A. phagocytophilum in domestic animals.

Characterization of reptile-associated Borrelia sp. in the vector tick, Amblyomma geoemydae, and its association with Lyme disease and relapsing fever Borrelia spp.

The genus Borrelia is arthropod-borne infectious agents in vertebrates, and is classified into Lyme disease (LD) Borrelia spp. and Relapsing fever (RF) Borrelia spp. In addition to these Borrelia groups, we recently reported reptile-associated (REP) Borrelia spp. from reptiles and from hard-bodied ticks, which probably transmitted the REP Borrelia spp. In this study, we investigated the presence of REP Borrelia sp. in moulted ticks, and found that trans-stadial transmission of REP Borrelia sp. occurred in the midgut, while it was observed that REP Borrelia sp. entered the salivary gland during blood-feeding. This characteristic is also found in LD Borrelia spp., which are also transmitted by hard-bodied ticks. Although phylogenetic analysis demonstrated that REP Borrelia spp. are similar to RF Borrelia spp., the ecology of the spirochaetes within the vector ticks is different for REP Borrelia spp. and RF Borrelia spp. Elucidation of the evolutionary history of the genus Borrelia and its adaptation to ticks promises to be of great interest to researchers of vector-borne microorganisms.

Ehrlichia chaffeensis Infection of Sika Deer, Japan

To determine whether Ehrlichia chaffeensis exists in Japan, we used PCR to examine blood from sika deer in Nara, Japan. Of 117 deer, 36 (31%) were infected with E. chaffeensis. The E. chaffeensis 16S rRNA base and GroEL amino acid sequences from Japan were most closely related to those of E. chaffeensis Arkansas.

Efficiency of pH-Sensitive Fusogenic Polymer-Modified Liposomes as a Vaccine Carrier

The usefulness of pH-sensitive fusogenic polymer-(succinylated poly(glycidol)-(SucPG-) modified liposomes as a vaccine carrier in the induction of immune responses was evaluated. Mice were intraperitoneally immunized with ovalbumin- (OVA-) containing SucPG-modified liposomes. After immunization, significant OVA-specific antibodies were detected in the serum. When sera were analyzed for isotype distribution, OVA-specific IgG1 antibody responses were noted in mice immunized with OVA-containing polymer-unmodified liposomes, whereas immunization with OVA-containing SucPG-modified liposomes resulted in the induction of OVA-specific IgG1, IgG2a, and IgG3 Ab responses. In spleen lymphocytes from mice immunized with OVA-containing SucPG-modified liposomes, both IFN-γ-(Th1-type-) and IL-4-(Th2 type-) specific mRNA were detected. Moreover, substantial production of IFN-γ and IL-4 was demonstrated in spleen cells from OVA-containing SucPG-modified liposomes in vitro. These results suggest that the pH-sensitive fusogenic polymer-(SucPG-) modified liposomes would serve effectively as an antigen delivery vehicle for inducing Th1 and Th2 immune responses.

A novel relapsing fever Borrelia sp. infects the salivary glands of the molted hard tick, Amblyomma geoemydae.

A novel relapsing fever Borrelia sp. was found in Amblyomma geoemydae in Japan. The novel Borrelia sp. was phylogenetically related to the hard (ixodid) tick-borne relapsing fever Borrelia spp. Borrelia miyamotoi and B. lonestari. The novel relapsing fever Borrelia sp. was detected in 39 A. geoemydae (39/274: 14.2%), of which 14 (14/274: 5.1%) were co-infected with the novel relapsing fever Borrelia sp. and Borrelia sp. tAG, one of the reptile-associated borreliae. Transstadial transmission of the novel relapsing fever Borrelia sp. occurred in the tick midgut and the salivary glands, although Borrelia sp. tAG was only detected in the tick midgut. The difference of the borrelial niche in molted ticks might be associated with borrelial characterization.