Molecular and antigenic comparison of Ehrlichia canis isolates from dogs, ticks, and a human in Venezuela

We previously culture isolated a strain of Ehrlichia canis, the causative agent of canine ehrlichiosis, from a human in Venezuela. In the present study, we examined whether dogs and ticks are infected with E. canis in Venezuela and, if so, whether this is the same strain as the human isolate. PCR analysis using E. canis-specific primers revealed that 17 of the 55 dog blood samples (31%) and all three pools of four Rhipicephalus sanguineus ticks each were positive. An ehrlichial agent (Venezuelan dog Ehrlichia [VDE]) was isolated and propagated in cell culture from one dog sample and was further analyzed to determine its molecular and antigenic characteristics. The 16S rRNA 1,408-bp sequence of the new VDE isolate was identical to that of the previously reported Venezuelan human Ehrlichia isolate (VHE) and was closely related (99.9%) to that of E. canis Oklahoma. The 5' (333-bp) and 3' (653-bp) sequences of the variable regions of the 16S rRNA genes from six additional E. canis-positive dog blood specimens and from three pooled-tick specimens were also identical to those of VHE. Western blot analysis of serum samples from three dogs infected with VDE by using several ehrlichial antigens revealed that the antigenic profile of the VDE was similar to the profiles of VHE and E. canis Oklahoma. Identical 16S rRNA gene sequences among ehrlichial organisms from dogs, ticks, and a human in the same geographic region in Venezuela and similar antigenic profiles between the dog and human isolates suggest that dogs serve as a reservoir of human E. canis infection and that R. sanguineus, which occasionally bites humans residing or traveling in this region, serves as a vector. This is the first report of culture isolation and antigenic characterization of an ehrlichial agent from a dog in South America, as well as the first molecular characterization of E. canis directly from naturally infected ticks.     

Topological analysis of a putative virB8 homologue essential for the cag type IV secretion system in Helicobacter pylori

The type IV secretion system encoded by the cag pathogenicity island of Helicobacter pylori is recognized as a major virulence determinant, governing the translocation of the CagA protein to eukaryotic cells. Despite the presence of 18 genes within the cag pathogenicity island which are essential for this process, several homologues encoding basic functions of a type IV secretion apparatus, such as a virB8 homologue, are still missing. We show here the presence of conserved VirB8 motifs in the cag-encoded apparatus protein HP530 and demonstrate that this protein adopts in E. coli a transmembrane topology similar to VirB8 and its homologues. Thus, we propose that HP530 is a type IV secretion component homologous to VirB8.     

Molecular detection of Ehrlichia canis,Anaplasma bovis,Anaplasma platys,Candidatus Midichloria mitochondrii and Babesia canis vogeli in ticks from Israel

Ticks are vectors of important pathogens of human and animals. Therefore, their microbial carriage capacity is constantly being investigated. The aim of this study was to characterize the diversity of domestic animal pathogens in ticks collected from vegetation and the ground, from different parts of Israel. Non-engorged questing adult ticks were collected from 13 localities. A total of 1196 ticks in 131 pools—83 pools of Rhipicephalus turanicus and 48 of Rhipicephalus sanguineus (with two to ten ticks per pool)—were included in this study. In addition, 13 single free-roaming Hyalomma spp. ticks were collected. Screening by molecular techniques revealed the presence of Ehrlichia canis, Anaplasma platys, Anaplasma bovis and Babesia canis vogeli DNA in R. turanicus ticks. E. canis, A. bovis, B. canis vogeli and Candidatus Midichloria mitochondrii DNA sequences were detected in R. sanguineus ticks. Candidatus Midichloria mitochondrii DNA was also detected in Hyalomma spp. ticks. Neither Hepatozoon spp. nor Bartonella spp. DNA was detected in any of the ticks examined. This study describes the first detection of E. canis in the tick R. turanicus, which may serve as a vector of this canine pathogen; E. canis was the most common pathogen detected in the collected questing ticks. It also describes the first detection of A. bovis and Candidatus Midichloria mitochondrii in Israel. To the best of the author's knowledge, this is the first report describing the detection of DNA of the latter two pathogens in R. sanguineus, and of A. bovis in R. turanicus.     

Brucella abortus virB12 is expressed during infection but is not an essential component of the type IV secretion system

The Brucella abortus virB operon, consisting of 11 genes, virB1 to virB11, and two putative genes, orf12 (virB12) and orf13, encodes a type IV secretion system (T4SS) that is required for intracellular replication and persistent infection in the mouse model. This study was undertaken to determine whether orf12 (virB12) encodes an essential part of the T4SS apparatus. The virB12 gene was found to encode a 17-kDa protein, which was detected in vitro in B. abortus grown to stationary phase. Mice infected with B. abortus 2308 produced an antibody response to the protein encoded by virB12, showing that this gene is expressed during infection. Expression of virB12 was not required for survival in J774 macrophages. VirB12 was also dispensable for the persistence of B. abortus, B. melitensis, and B. suis in mice up to 4 weeks after infection, since deletion mutants lacking virB12 were recovered from splenic tissue at wild-type levels. These results show that VirB12 is not essential for the persistence of the human-pathogenic Brucella spp. in the mouse and macrophage models of infection.     

Prevalence of Ehrlichia canis (Rickettsiales: Anaplasmataceae) in dogs and Rhipicephalus sanguineus (Acari: Ixodidae) ticks from Brazil

The current study evaluated the prevalence of Ehrlichia canis Donatien and Lestoquard in domestic dogs, Canis familiaris L., and Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae) ticks from different areas of Brazil. In Monte Negro County (state of Rond?nia, Brazilian western Amazon), the indirect immunofluorescence assay detected E. canis-reactive antibodies (titer > or = 40) in 58/153 (37.9%) and 40/161 (24.8%) dogs from the urban and rural areas, respectively. These values were significantly different between the two areas. Ticks from a household in the urban area of Monte Negro, and from households in three other localities (162-165 adult ticks per household) in the state of S?o Paulo (SP) were tested by polymerase chain reaction (PCR) targeting an erlichial dsb gene fragment. The prevalence of infected ticks (given as minimal infection rate) was 2.3, 6.2, and 3.7% for populations 1 (Monte Negro), 2 (Jundiaí, SP), and 3 (S?o Paulo I, SP), respectively, which were statistically similar. In contrast, no infected tick was detected in population 4 (S?o Paulo II, SP). DNA sequences were determined for some of the PCR products generated from ticks and dogs from populations 1-3, being all identical to each other and to available sequences of E. canis in GenBank. These results reinforce previous records of E. canis-infecting dogs in Brazil. Natural infection of R. sanguineus ticks by E. canis is reported for the first time in Brazil, where this tick is the commonest species infesting dogs.     

Prevalence and comparative analysis of the type IV secretion system in Aggregatibacter actinomycetemcomitan

Backgroud/purpose

Aggregatibacter actinomycetemcomitans has emerged as one of the aetiological agents in periodontal disease. Although Type IV secretion systems (T4SSs) are widely distributed in many bacteria, the genetic features and distribution of T4SSs in A. actinomycetemcomitans remain unclear. In this study, we investigated the prevalence of A. actinomycetemcomitans serotypes and their T4SSs in a Taiwanese population.

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.     

Western immunoblotting analysis of the antibody responses of patients with human monocytotropic ehrlichiosis to different strains of Ehrlichia chaffeensis and Ehrlichia canis.

In order to evaluate the relative sensitivity of the detection of antibodies against various antigenic proteins of Ehrlichia chaffeensis for the diagnosis of the emerging infectious disease human monocytotropic ehrlichiosis, Western immunoblotting was performed with 27 serum samples from convalescent patients with antibodies, as demonstrated by indirect immunofluorescence assay. Among 22 patients with antibodies reactive with the 120-kDa protein, 15 showed reactivity with the 29/28-kDa protein(s) and the proteins in the 44- to 88-kDa range. Two of the serum samples with this pattern reacted with the 29/28-kDa protein(s) of only the 91HE17 strain, and one sample reacted with only that of the Arkansas strain, indicating that the antibodies were stimulated by strain-specific epitopes. Overall, antibodies to the 29/28-kDa protein(s) were detected in only 16 patients' sera, suggesting that this protein is less sensitive than the 120-kDa protein. Two of 12 serum samples from healthy blood donors had antibodies reactive with the 120-kDa protein; one of these samples reacted also with the 29/28-kDa protein(s) of Ehrlichia canis, suggesting that unrecognized ehrlichial infection might have occurred, including human infection with E. canis. A high correlation between reactivity with the 120-kDa protein by Western immunoblotting and the recombinant 120-kDa protein by dot blot supports the potential usefulness of this recombinant antigen in diagnostic serology.     

Genetic and antigenic diversities of major immunoreactive proteins in globally distributed Ehrlichia canis strains

The extent of knowledge regarding the diversity of globally distributed Ehrlichia canis strains has been limited to information gained from a few evolutionarily conserved genes. In this study, E. canis strains from the United States (strain Jake [US]), Brazil (strain São Paulo [BR]), and Israel (strain 611 [IS] and Ranana [IS-R]) were used to examine the antigenic and genetic diversities of four well-characterized major immunoreactive protein genes/proteins. gp36 and gp200 were the most divergent genes, and nucleotide substitutions in the gp36 tandem repeat region of the IS strain, but not the IS-R strain, resulted in two amino acid differences (S→P and P→T) in each nine-amino-acid repeat (epitope-containing region). DNA sequences of gp19 and gp140 were completely conserved in the US and BR strains, but differences were found in the Israeli strains, including two fewer tandem repeats in gp140 and a single amino acid substitution in gp19 from the IS strain. E. canis whole-cell lysates from each isolate were examined by Western immunoblotting using sera from naturally infected dogs from each country, and four major immunoreactive proteins (gp19, gp36, gp140, and gp200) were identified in each strain using protein-specific antisera. The US and BR strains exhibited highly conserved immunoreactive protein profiles, while some differences were identified in the IS strain. Sera from naturally infected Israeli dogs confirmed gene sequencing information, which demonstrated two distinct E. canis strains, defined by the gp36 gene. Conversely, gp19 was strongly reactive and present in all E. canis isolates. gp140 and gp200 were also present in all strains, although gp140 in the IS strain had two fewer tandem repeats and exhibited a smaller mass.Ehrlichia canis is a globally distributed, tick-transmitted, obligately intracellular bacterium that is the primary etiological agent of canine monocytic ehrlichiosis and has been identified as being the cause of human ehrlichiosis in patients from Venezuela (38, 39). Rickettsiosis in dogs caused by E. canis was first reported in 1935 in Algeria and was later reported in southern India and other parts of Africa in the 1940s (9, 31). Subsequently, E. canis was relatively unrecognized until it was associated with outbreaks of canine tropical pancytopenia in Singapore and Malaysia from 1963 to 1968 (51) and was identified as being the cause of an epizootic of canine tropical pancytopenia in U.S. military dogs stationed in Vietnam in late 1968 (17, 36). E. canis infections have since been well documented in the United States, Israel, Brazil, and Vietnam (1, 3, 12, 16, 20-22, 36, 49), and serologic and/or molecular evidence of infection in temperate regions where Rhipicephalus sanguineus is commonly found, including Central and South America, the Caribbean, parts of Africa, southern Europe, and southeast Asia, has also been reported (2, 5-8, 15, 18, 19, 23, 32, 33, 41, 42, 44, 50).The development of globally useful serologically and molecularly based diagnostics as well as effective vaccines for canine monocytic ehrlichiosis is dependent on an understanding of the genetic diversity of E. canis, particularly with respect to major immunoreactive proteins. Molecular characterization of evolutionarily conserved genes such as 16S rRNA has provided little information on strain diversity and suggests a high level of conservation (39, 40, 43, 47, 48). Similarly, the immunoreactive major outer membrane proteins p28 and p30 in U.S. and Venezuelan strains of E. canis appear to be highly conserved (13, 29, 30, 46), an observation that was extended to characterized E. canis strains from six human patients from Venezuela (38). Other genes such as the thio-oxidoreductase gene (dsb) and gltA were also found to be conserved in geographically dispersed strains (23, 32).The genome of E. canis has been sequenced, and a small group of acidic tandem repeat- and ankyrin repeat-containing proteins associated with host-pathogen interactions were identified (24). Several of these proteins are considered major immunoreactive proteins and have been well studied, including gp200, gp140, gp36, and gp19 (11, 25, 26, 28, 53). E. canis gp36 is an acidic serine-rich protein that contains a major antibody epitope in the tandem repeat region (11). Examination of the gp36 gene in U.S., Brazilian, and Cameroonian strains of E. canis identified variations in the numbers of tandem repeats and nucleic acid changes that resulted in four amino acid substitutions (10). However, the diversities of other major immunoreactive E. canis proteins in globally dispersed strains are not known. A homogeneous pattern of proteins reacting with E. canis dog sera from the United States, France, Israel, and the Virgin Islands by immunoblotting was previously reported (14). However, differences in protein reactivity were noted with sera collected from dogs from Italy and Zimbabwe, suggesting the potential for diversity in the antigenic composition of E. canis strains in these countries (14).The objective of this study was to determine the genetic and antigenic diversities of proteins subject to immune pressure in globally dispersed strains of E. canis. Four major immunoreactive protein genes (gp200, gp140, gp36, and gp19) were sequenced from each strain, and immunoblotting profiles for E. canis whole-cell lysates were compared. Strains from the United States and Brazil exhibited homogeneous immunoblotting patterns compared to that of the strain from Israel. Sequencing of four major immunoreactive protein genes demonstrated that U.S. and Brazilian strains were highly similar and that strains from Israel were the more divergent.     

Detection of Anaplasma phagocytophilum and Ehrlichia sp. HF strains in Ixodes ricinus ticks in Brittany, France

DNA extracts from 156 tick pools, 18 blood specimens and 17 spleens from European woodmice (Apodemus sylvaticus) collected in Brittany, France were tested by PCR for the 16S rRNA gene of Anaplasmataceae. Positive amplicons were sequenced and confirmed, either by amplification and sequencing of a second gene, or by a second PCR specific for the P44 and gltA genes of Anaplasma phagocytophilum and the gltA gene of Ehrlichia sp. HF. In addition to A. phagocytophilum, the study detected Ehrlichia sp. HF for the first time in Ixodes ricinus ticks. This organism has only been detected previously in Ixodes ovatus ticks from Japan.     

Detection of Ehrlichia canis in canine carrier blood and in individual experimentally infected ticks with a p30-based PCR assay

Detection of vector-borne pathogens is necessary for investigation of their association with vertebrate and invertebrate hosts. The ability to detect Ehrlichia spp. within individual experimentally infected ticks would be valuable for studies to evaluate the relative competence of different vector species and transmission scenarios. The purpose of this study was to develop a sensitive PCR assay based on oligonucleotide sequences from the unique Ehrlichia canis gene, p30, to facilitate studies that require monitoring this pathogen in canine and tick hosts during experimental transmission. Homologous sequences for Ehrlichia chaffeensis p28 were compared to sequences of primers derived from a sequence conserved among E. canis isolates. Criteria for primer selection included annealing scores, identity of the primers to homologous E. chaffeensis sequences, and the availability of similarly optimal primers that were nested within the target template sequence. The p30-based assay was at least 100-fold more sensitive than a previously reported nested 16S ribosomal DNA (rDNA)-based assay and did not amplify the 200-bp target amplicon from E. chaffeensis, the human granulocytic ehrlichiosis agent, or Ehrlichia muris DNA. The assay was used to detect E. canis in canine carrier blood and in experimentally infected Rhipicephalus sanguineus ticks. Optimized procedures for preparing tissues from these hosts for PCR assay are described. Our results indicated that this p30-based PCR assay will be useful for experimental investigations, that it has potential as a routine test, and that this approach to PCR assay design may be applicable to other pathogens that occur at low levels in affected hosts.     

Association and evidence for linked recognition of type IV secretion system proteins VirB9-1, VirB9-2, and VirB10 in Anaplasma marginale

Like several other bacterial pathogens, Anaplasma marginale has an outer membrane that induces complete protection from infection and disease. However, the proteins that confer protective immunity and whether protection requires interacting proteins and/or linked T-cell and immunoglobulin G epitopes are not known. Our goal is to target the conserved type IV secretion system (T4SS) to identify conserved, immunogenic membrane proteins that are interacting and linked recognition candidates. Linked recognition is a process by which a B cell is optimally activated by a helper T cell that responds to the same, or physically associated, antigen. A. marginale T4SS proteins VirB2, VirB4-1, VirB4-2, VirB6-1, VirB7, VirB8-2, VirB9-1, VirB9-2, VirB10, VirB11, and VirD4 were screened for their ability to induce IgG and to stimulate CD4+ T cells from outer membrane-vaccinated cattle. VirB9-1, VirB9-2, and VirB10 induced the strongest IgG and T-cell responses in the majority of cattle, although three animals with major histocompatibility complex class II DRB3 restriction fragment length polymorphism types 8/23, 3/16, and 16/27 lacked T-cell responses to VirB9-1, VirB9-1 and VirB9-2, or VirB9-2 and VirB10, respectively. For these animals, VirB9-1-, VirB9-2-, and VirB10-specific IgG production may be associated with T-cell help provided by responses to an interacting protein partner(s). Interacting protein partners indicated by far-Western blotting were confirmed by immunoprecipitation assays and revealed, for the first time, specific interactions of VirB9-1 with VirB9-2 and VirB10. The immunogenicity and interactions of VirB9-1, VirB9-2, and VirB10 justify their testing as a linked protein vaccine against A. marginale.     

转染Smad 2基因的大鼠肾系膜细胞纤连蛋白和Ⅳ型胶原表达的改变

目的通过对大鼠肾小球系膜细胞(MsC)转染Smad 2基因,观察转染的阳性细胞克隆纤连蛋白(FN)及Ⅳ型胶原(ColⅣ)表达的改变,以探讨转化生长因子β(TGF-β)/Smad信号转导通路引起细胞外基质积聚导致肾小球硬化发生的分子机制.方法经磷酸钙介导将含有Smad 2重组表达质粒转染大鼠MsC,用G418筛选及Western印迹分析法鉴定,又分别采用Western印迹分析法和逆转录-聚合酶链反应(RT-PCR)检测转染阳性细胞克隆FN及ColⅣ表达的改变.结果成功建立高表达Smad 2蛋白的阳性MsC克隆(T-12、T-31、T-35、T-40),并证实其FN及ColⅣ的mRNA和蛋白的表达水平均明显上调.阳性MsC克隆FN蛋白为对照组2.4倍(P<0.05),mRNA为对照组的2.7倍(P<0.05);阳性MsC克隆ColⅣ蛋白为对照组2.9 倍(P<0.01);mRNA为对照组的3.3倍(P<0.01 ). 结论 TGF-β/Smad信号转导途径中的Smad 2可能是促进FN及ColⅣ在肾小球中积聚,是导致肾小球硬化发生的重要信号转导分子.     

转染结缔组织生长因子基因的大鼠肾系膜细胞纤连蛋白和Ⅳ型胶原表达的改变

目的观察大鼠肾系膜细胞（MsC）转染结缔组织生长因子（CTGF）基因转染阳性细胞克隆中纤连蛋白（FN）和Ⅳ型胶原（ColⅣ）表达的改变,探讨CTGF在肾小球硬化发生中的作用。方法经脂质体介导将含有CTGF的重组表达质粒转染至大鼠MsC,用G418筛选及Western blot和半定量逆转录聚合酶链反应作鉴定;检测阳性克隆FN、ColⅣ蛋白及其mRNA表达的改变。结果成功建立高表达CTGF的阳性MsC克隆（MCT-1,2）,并证实其与对照组相比,阳性MsC克隆FN、ColⅣ的表达均有上调,其FN蛋白及其mRNA表达分别增高2．9倍和3．2倍,ColⅣ蛋白和mRNA表达分别增高为2．4倍和3．8倍。结论CTGF可促进MsC的FN、ColⅣ的表达。表明其在肾小球硬化发生中起促进作用。     

Interferon-gamma stimulates the expression of galectin-9 in cultured human endothelial cells

Galectin-9 is a member of the galectin family and has been identified as an eosinophil chemoattractant produced by activated T lymphocytes. Vascular endothelial cells play an important role in the initial step of eosinophil recruitment and activation in immune and inflammatory responses. We have addressed the stimulation of galectin-9 expression in endothelial cells. Galectin-9 was detected in membrane and cytosolic fractions of human umbilical vein endothelial cells stimulated with interferon-gamma (IFN-gamma). IFN-gamma also enhanced the adhesion of human eosinophilic leukemia-1 cells to endothelial monolayers, and it was inhibited by the presence of lactose. Interleukin-4, which induces eotaxin expression, did not affect the expression of galectin-9. The in situ endothelium from patients with inflammatory diseases was found to express galectin-9. IFN-gamma-induced production of galectin-9 by endothelial cells may play an important role in immune responses by regulating interactions between the vascular wall and eosinophils.     

Zoonotic Anaplasma phagocytophilum,Ehrlichia canis,Dirofilaria immitis,Borrelia burgdorferi,and spotted fever group rickettsiae (SFGR) in different types of dogs

Parasitology Research - Dogs can carry and share zoonotic pathogens with humans. This problem is understudied in different parts of the world, including Jordan. This study determined the prevalence...     

Altered secretion of type III procollagen in a form of type IV Ehlers-Danlos syndrome. Biochemical studies in cultured fibroblasts

Cultured dermal fibroblasts from a woman with one variety of type IV Ehlers-Danlos syndrome synthesized type III procollagen but fail to secrete the bulk of the protein. Although total collagen production is similar to that of controls, the affected cells retain almost twice as much collagen as controls. The additional retained protein is a disulfide-boned collagenous trimer that remains disulfide-linked after limited proteolysis with pepsin and, after pepsin treatment, migrates with type III collagen on polyacrylamide gel electrophoresis. Affected cells have markedly increased staining with antibodies directed against type III procollagen. These studies indicate that decreased secretion of type III procollagen that is synthesized can result in the clinical syndrome of type IV Ehlers-Danlos syndrome.