In vitro establishment and propagation of a Brazilian strain of Anaplasma marginale with appendage in IDE8 (Ixodes scapularis) cells.
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ABSTRACT:
SUBMITTER: Bastos CV
PROVIDER: S-EPMC3769711 | BioStudies | 2009-01-01
REPOSITORIES: biostudies
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Project description:Anaplasma marginale, the causative agent of bovine anaplasmosis, is a tick-borne bacterium that causes significant economic losses for cattle industries and is increasingly being detected in other animal species. Rhipicephalus microplus is the main vector of this bacterium and may be found parasitizing small ruminants. In northeastern Brazil, multispecies grazing is a common family subsistence practice on smallholder farms possibly facilitating interspecies transmission of pathogens. Considering that A. marginale infection has been previously molecularly described in sheep, this study has aimed to estimate the prevalence of A. marginale and factors associated with the infection in goats from northeastern Brazil. A total of 403 goat blood samples were included in the study. An epidemiological questionnaire was applied to each farm owner addressing age, gender, presence of ticks and multispecies grazing. All samples were screened for A. marginale- and A. ovis-infection using primers targeting the Anaplasma spp. msp4 gene. The identity of A. marginale in the blood was confirmed by PCR amplification of msp5 followed by sequencing. Anaplasma spp. were differentiated by sequencing of the repeat region of the msp1? gene. For the statistical analysis the Chi-square or the Fisher's exact test was used to verify association of the individual factors (age, gender, presence of ticks, and multispecies grazing) with Anaplasma spp. infection. We report the first molecular detection of A. marginale in goats from northeastern Brazil, based on msp1?, msp4 and msp5 gene sequencing analysis. Sequencing of the detected A. marginale msp1? gene revealed the F repeat. Amblyomma parvum and R. microplus were found feeding on animals.
2018-01-01 | S-EPMC6089441 | BioStudies
Project description:Bacterial pathogens in the genera Anaplasma and Ehrlichia encode a protein superfamily, pfam01617, which includes the predominant outer membrane proteins (OMPs) of each species, major surface protein 2 (MSP2) and MSP3 of Anaplasma marginale and Anaplasma ovis, Anaplasma phagocytophilum MSP2 (p44), Ehrlichia chaffeensis p28-OMP, Ehrlichia canis p30, and Ehrlichia ruminantium MAP1, and has been shown to be involved in both antigenic variation within the mammalian host and differential expression between the mammalian and arthropod hosts. Recently, complete sequencing of the A. marginale genome has identified an expanded set of genes, designated omp1-14, encoding new members of this superfamily. Transcriptional analysis indicated that, with the exception of the three smallest open reading frames, omp2, omp3, and omp6, these superfamily genes are transcribed in A. marginale-infected erythrocytes, tick midgut and salivary glands, and the IDE8 tick cell line. OMPs 1, 4, 7 to 9, and 11 were confirmed to be expressed as proteins by A. marginale within infected erythrocytes, with expression being either markedly lower (OMPs 1, 4, and 7 to 9) or absent (OMP11) in infected tick cells, which reflected regulation at the transcript level. Although the pfam01617 superfamily includes the antigenically variable MSP2 and MSP3 surface proteins, analysis of the omp1-14 sequences throughout a cycle of acute and persistent infection in the mammalian host and tick transmission reveals a high degree of conservation, an observation supported by sequence comparisons between the St. Maries strain and Florida strain genomes.
2006-01-01 | S-EPMC1479288 | BioStudies
Project description:Anaplasma marginale, the causative agent of bovine anaplasmosis, is a tick-borne rickettsial pathogen of cattle that multiplies in erythrocytes and tick cells. Major surface protein 1a (MSP1a) and MSP1b form the MSP1 complex of A. marginale, which is involved in adhesion of the pathogen to host cells. In this study we tested the hypothesis that MSP1a and MSP1b were glycosylated, because the observed molecular weights of both proteins were greater than the deduced molecular masses. We further hypothesized that the glycosylation of MSP1a plays a role in adhesion of A. marginale to tick cells. Native and Escherichia coli-derived recombinant MSP1a and MSP1b proteins were shown by gas chromatography to be glycosylated and to contain neutral sugars. Glycosylation of MSP1a appeared to be mainly O-linked to Ser/Thr residues in the N-terminal repeated peptides. Glycosylation may play a role in adhesion of A. marginale to tick cells because chemical deglycosylation of MSP1a significantly reduced its adhesive properties. Although the MSP1a polypeptide backbone alone was adherent to tick cell extract, the glycans in the N-terminal repeats appeared to enhance binding and may cooperatively interact with one or more surface molecules on host cells. These results demonstrated that MSP1a and MSP1b are glycosylated and suggest that the glycosylation of MSP1a plays a role in the adhesion of A. marginale to tick cells.
2004-01-01 | S-EPMC387886 | BioStudies
Project description:BACKGROUND:Anaplasma marginale is an obligate intracellular bacterium and the main cause of bovine anaplasmosis in tropical and subtropical regions. In Egypt, data regarding the prevalence of A. marginale in ruminant hosts and of the circulating genotypes is lacking. This study therefore aimed to (i) investigate the presence, epidemiology and genotypes of A. marginale in cattle and buffaloes in Egypt, (ii) to evaluate suitable diagnostic tools and (iii) to identify co-infections of A. marginale with other selected tick-borne pathogens. METHODS:Blood samples were collected from 394 animals (309 cattle and 85 buffaloes) from three different areas in Egypt. For the detection of A. marginale infection, several tests were compared for their sensitivity and specificity: blood smear analysis, enzyme-linked immunosorbent assay (ELISA), PCR, real-time PCR and reverse line blot (RLB) assay. Co-infections with A. marginale, piroplasms and other Anaplasmataceae were surveyed by RLB while A. marginale genotypes were identified by amplifying and sequencing the partial msp1? gene. RESULTS:Anaplasma marginale DNA was amplified by qPCR in 68.3% of cattle and 29.4% of buffaloes. RLB showed infection with A. marginale in 50.2% of cattle and 42.5% of buffaloes. Blood smear analysis detected this agent in 16.2% of cattle and 2.4% of buffaloes. ELISA showed specific antibodies against A. marginale in 54.9% of cattle. Anaplasma marginale was associated, in cattle and buffaloes, with several tick-borne pathogens (Theileria annulata, Babesia bovis, Babesia bigemina, Babesia occultans and Anaplasma platys). A significant difference of A. marginale infection level was noticed in cattle, where animals between 3-5-years-old had a higher prevalence (79.2%) compared to those older than 5 years (36.4%) and younger than 3 years (59.7%) and one year (64.5%), respectively (P?=?0.002281). Microsatellite analysis identified 15 different genotypes. CONCLUSIONS:The epidemiological findings revealed high prevalence of A. marginale in cattle and buffaloes in all the investigated areas. The circulation of diverse genotypes was observed, most of these A. marginale genotypes being specific for Egypt. The qPCR assay was confirmed to be the most sensitive tool for detection of A. marginale in cattle and buffaloes even in the carrier state, highlighting the importance of using suitable diagnostic tests.
2020-01-01 | S-EPMC7526245 | BioStudies
Project description:Anaplasma marginale, a tick-borne rickettsial pathogen of cattle, is endemic in several areas of the United States. Many geographic isolates of A. marginale that occur in the United States are characterized by the major surface protein 1a, which varies in sequence and molecular weight due to different numbers of tandem repeats of 28 or 29 amino acids. Recent studies (G. H. Palmer, F. R. Rurangirwa, and T. F. McElwain, J. Clin. Microbiol. 39:631-635, 2001) of an A. marginale-infected herd of cattle in an area of endemicity demonstrated that multiple msp1alpha genotypes were present but that only one genotype was found per individual bovine. These findings suggested that infection of cattle with other genotypes was excluded. The present study was undertaken to confirm the phenomenon of infection exclusion of A. marginale genotypes in infected bovine erythrocytes and cultured tick cells. Two tick-transmissible isolates of A. marginale, one from Virginia and one from Oklahoma, were used for these studies. In two separate trials, cattle inoculated with equal doses of the two isolates developed infection with only one genotype. Tick cell cultures inoculated with equal doses of the two isolates became infected with only the Virginia isolate of A. marginale. When cultures were inoculated with different ratios of the Oklahoma and Virginia isolates of A. marginale, the isolate inoculated in the higher ratio became established and excluded infection with the other. When cultures with established infections of one isolate were subsequently infected with the other, only the established isolate was detected. We documented infection exclusion during initial infection in cell culture by labeling each isolate with a different fluorescent dye. After 2 days in culture, only a single isolate was detected per cell by fluorescence microscopy. Finally, when Anaplasma ovis infections were established in cultures that were subsequently inoculated with the Virginia or Oklahoma isolate of A. marginale, A. marginale infection was excluded. These studies confirm that infection exclusion occurs with A. marginale in bovine erythrocytes and tick cells, resulting in the establishment of only one genotype, and appears to be the first report of infection exclusion for Anaplasma and Ehrlichia species.
2002-01-01 | S-EPMC119989 | BioStudies
Project description:Anaplasma marginale is the causative agent of bovine anaplasmosis, a disease of worldwide economic importance. Major surface proteins (MSPs) are involved in host-pathogen and tick-pathogen interactions and they have been used as markers for the genetic characterization of A. marginale strains and phylogenetic studies. The major surface protein 5 (MSP5) is highly conserved in the genus Anaplasma and in all isolates of A. marginale. The aim of the present work was to carry out the cloning, sequencing and characterization of the recombinant MSP5 Anaplasma marginale Havana isolate. The sequence of the msp5 gene of Anaplasma marginale Havana isolate with a size of 633 pb was determined (Acc. No. AY527217). This gene was cloned into pRSETB vector and expressed in Escherichia coli. The MSP5 protein was recognized by the monoclonal antibody ANAF16C1 and it showed a high similitude percent with the gene sequence described for other Anaplasma marginale isolates. These data are very important for the development of a diagnostic test for A. marginale using the MSP5 recombinant protein.
2009-01-01 | S-EPMC3768588 | BioStudies
Project description:The rickettsia Anaplasma marginale is the most prevalent tick-borne livestock pathogen worldwide and is a severe constraint to animal health. A. marginale establishes lifelong persistence in infected ruminants and these animals serve as a reservoir for ticks to acquire and transmit the pathogen. Within the mammalian host, A. marginale generates antigenic variants by changing a surface coat composed of numerous proteins. By sequencing and annotating the complete 1,197,687-bp genome of the St. Maries strain of A. marginale, we show that this surface coat is dominated by two families containing immunodominant proteins: the msp2 superfamily and the msp1 superfamily. Of the 949 annotated coding sequences, just 62 are predicted to be outer membrane proteins, and of these, 49 belong to one of these two superfamilies. The genome contains unusual functional pseudogenes that belong to the msp2 superfamily and play an integral role in surface coat antigenic variation, and are thus distinctly different from pseudogenes described as byproducts of reductive evolution in other Rickettsiales.
2005-01-01 | S-EPMC545514 | BioStudies
Project description:Anaplasma marginale (Rickettsiales: Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world. Although serologic tests have identified American bison, Bison bison, as being infected with A. marginale, the present study was undertaken to confirm A. marginale infection and to characterize isolates obtained from naturally infected bison in the United States and Canada. Major surface protein (MSP1a and MSP4) sequences of bison isolates were characterized in comparison with New World cattle isolates. Blood from one U.S. bison was inoculated into a susceptible, splenectomized calf, which developed acute anaplasmosis, demonstrating infectivity of this A. marginale bison isolate for cattle. The results of this study showed that these A. marginale isolates obtained from bison were similar to ones from naturally infected cattle.
2003-01-01 | S-EPMC169112 | BioStudies
Project description:BACKGROUND: Bovine anaplasmosis has been reported in several European countries, but the vector competency of tick species for Anaplasma marginale from these localities has not been determined. Because of the wide distributional range of Dermacentor reticulatus within Europe and the major role of Dermacentor spp. as a vector of A. marginale in the United States, we tested the vector competency of D. reticulatus for A. marginale. RESULTS: Male D. reticulatus were allowed to feed for 7 days on a calf persistently infected with a Zaria isolate of A. marginale, after which they were removed and held off-host for 7 days. The ticks were then allowed to feed a second time for 7 days on a susceptible tick-naïve calf. Infection of calf No. 4291 was detected 20 days post exposure (p.i.) and confirmed by msp4 PCR. Thirty percent of the dissected acquisition fed ticks was infected. In addition, A. marginale colonies were detected by light microscopy in the salivary glands of the acquisition fed ticks. Transmission of A. marginale to calf No. 9191 was confirmed by examination of Giemsa-stained blood smears and msp4 PCR. Ticks were dissected after transmission feeding and presence of A. marginale was confirmed in 18.5% of the dissected ticks. CONCLUSION: This study demonstrates that D. reticulatus males are competent vectors of A. marginale. Further studies are needed to confirm the vector competency of D. reticulatus for other A. marginale strains from geographic areas in Europe.
2007-01-01 | S-EPMC2217534 | BioStudies
Project description:A gene for the beta subunit of the immunoprotective surface antigen MSP-1 of Anaplasma marginale was previously cloned and expressed in Escherichia coli. A nucleic acid probe based on this gene detects A. marginale infection in carrier cattle and in the tick vector. We report here the sequence and structural features of the cloned msp1 beta gene and expressed polypeptide. The gene codes for a polypeptide of 756 amino acids that contains domains of tandemly repeated sequence and glutamine-rich regions at the N and C termini. The cloned copy is a member of a multigene family with multiple restriction fragment length polymorphisms in isolates of this rickettsia from different geographical regions. The availability of the sequence will allow use of the polymerase chain reaction in diagnostic assays and the preparation and testing of different vaccine constructs in cattle.
1000-01-01 | S-EPMC258354 | BioStudies