Major Surface Protein 1a Research Articles

Characterization of strains of Anaplasma marginale from clinical cases in bovine using major surface protein 1a in Uruguay.

The major surface protein 1a (MSP1a) gene has been used to characterize Anaplasma marginale genetic diversity. This pathogen causes significant productivity and economic losses to the cattle industry. The objective of the present study was to report the first characterization of A. marginale genetic diversity in Uruguay based on MSP1a genotypes and their putative relationship with Rhipicephalus microplus. This cross-sectional study was conducted between 2016 and 2020. The study included whole blood samples from clinical cases of bovine anaplasmosis obtained from 30 outbreaks located in six Uruguay territorial departments. Diagnosis was performed using Giemsa-stained smears and confirmed by nested Polymerase Chance Reaction (nPCR) targeting the A. marginale major surface protein 5 gene. The genetic diversity of A. marginale strains was characterized by analyzing the microsatellite and tandem repeats of MSP1a. Based on the microsatellite structure, four genotypes were identified. Genotype E was the most prevalent. Analysis of MSP1a tandem repeats showed 28 different strains from the combination of 31 repeats, with τ-10-15 and α-β-β-β-Γ being the most common. Repeats Γ, β, α, and γ were associated with the absence of R. microplus with statistical significance (p < 0.05). Molecular observations showed that 46.7% of the strains identified in our samples lacked the ability to bind to tick cells; therefore, they were probably transmitted by other vectors. Strain genetic diversity provides valuable information for understanding the epidemiological behavior of A. marginale and could contribute to the development of effective vaccines for the control of this disease.

PSV-1 A Subcutaneous Ear Implant to Deliver an Anaplasmosis Vaccine to Dairy Steers

Abstract Bovine anaplasmosis is the most prevalent tick-transmitted disease of cattle worldwide and a major obstacle to beef production. The objective of the study was to test the ability of a single-dose delivery platform to produce long-lasting immunity against anaplasmosis infections. Twelve Holstein steers were administered a 3-stage, single-dose vaccine against Anaplasma marginale major surface protein 1a. The vaccine consisted of a soluble vaccine administered subcutaneously for immune priming, a biodegradable polyanhydride depot for intermediate slow-release of the vaccine for boosting the immune response, and an immune-isolated vaccine platform for extended antigen release deposited subcutaneously in the ear. Six calves were randomly assigned to two vaccine constructs that featured rods and implants containing a combination of two adjuvants (DEAE-Dextran and Quil-A). The remaining 6 calves were randomly assigned to two vaccine constructs that featured rods and implants containing the same adjuvant (DEAE-Dextran or Quil-A). Twenty months post-implantation, calves were challenged with Anaplasma marginale stabilate and were monitored weekly for signs of fever, decreased packed cell volume (PCV) and bacteremia. Outcome variables (fever, PCV, bacteremia) were analyzed using a linear mixed model (PROC GLMMIX; SAS University Edition v9.04.01, SAS Institute, Cary, NC). Two-tailed chi-squared tests were used to compare need for antibiotic intervention between vaccine constructs. Calves within the combination adjuvant construct group (DEAE-D and Quil-A) exhibited significantly (P = .006) higher PCV than calves within the single adjuvant construct group (DEAE-Dextran or Quil-A). Likewise, calves within the combination adjuvant group required significantly (P = .014) fewer antibiotic interventions compared with calves in the single adjuvant group. Findings of this study suggest that calves exhibited significantly diminished clinical signs of anaplasmosis when vaccine was delivered with a combination of adjuvants as opposed to a single adjuvant in a single-dose delivery platform.

Genetic diversity of major surface protein 1a of Anaplasma marginale in dairy cattle.

Major surface protein 1 alpha (Msp1α) is a stable genetic marker for identifying Anaplasma marginale. Our aims were to investigate the genetic diversity of A. marginale based on microsatellites and tandem repeats of the msp1α gene and to determine the phylogenetic relationships among six isolates obtained from 63 dairy cows positive for the pathogen from Turkey and from strains worldwide. Msp1α microsatellite analysis revealed infection with three genotypes (B, C, G). Genotype B was found in cows held in an intensive management system, while genotypes C and G were found in grazing cows. Microsatellite sequences produced SD-ATG distances of 19 and 23 nucleotides, with 23 nucleotides predominating, showing high infection potential. Fourteen different tandem repeats of A. marginale were found in the samples, and four msp1α gene repeats designated Tr4, Tr5, Tr6 and Tr7 were newly described. The majority of A. marginale isolates exhibited more than three Msp1a tandem repeats, with a maximum of seven. The Msp1a tandem repeats α-73-73-73-73-73-73 were commonly observed in intensively managed cows. The phylogenetic analysis revealed A. marginale strains from Turkey clustering with those of the Americas, Africa, Asia and Europe, suggesting a history of cattle trade and transport.

Development of a subcutaneous ear implant to deliver an anaplasmosis vaccine to dairy steers.

Bovine anaplasmosis is the most prevalent tick-transmitted disease of cattle worldwide and a major obstacle to profitable beef production. Use of chlortetracycline-medicated feed to control active anaplasmosis infections during the vector season has raised concerns about the potential emergence of antimicrobial resistance in bacteria that may pose a risk to human health. Furthermore, the absence of effectiveness data for a commercially available, conditionally licensed anaplasmosis vaccine is a major impediment to implementing anaplasmosis control programs. The primary objective of this study was to develop a single-dose vaccine delivery platform to produce long-lasting protective immunity against anaplasmosis infections. Twelve Holstein steers, aged 11 to 12 wk, were administered a novel 3-stage, single-dose vaccine against Anaplasma marginale, a major surface protein 1a. The vaccine consisted of a soluble vaccine administered subcutaneously (s.c.) for immune priming, a vaccine depot of a biodegradable polyanhydride rod with intermediate slow release of the vaccine for boosting immune response, and an immune-isolated vaccine platform for extended antigen release (VPEAR implant) deposited s.c. in the ear. Six calves were randomly assigned to 2 vaccine constructs (n = 3) that featured rods and implants containing a combination of 2 different adjuvants, diethylaminoethyl (DEAE)-Dextran and Quil-A (Group A). The remaining 6 calves were randomly assigned to 2 vaccine constructs (n = 3) that featured rods and implants containing the same adjuvant (either DEAE-Dextran or Quil A) (Group B). Twenty-one months post-implantation, calves were challenged intravenously with A. marginale stabilate and were monitored weekly for signs of fever, decreased packed cell volume (PCV) and bacteremia. Data were analyzed using a mixed-effects model and chi-squared tests (SAS v9.04.01, SAS Institute, Cary, NC). Calves in Group A had higher PCV than calves in Group B (P = 0.006) at day 35 post-infection. Calves in Group A were less likely to require antibiotic intervention compared with calves in Group B (P = 0.014). Results indicate that calves exhibited diminished clinical signs of anaplasmosis when antigen was delivered with a combination of adjuvants as opposed to a single adjuvant. This demonstrates the feasibility of providing long-lasting protection against clinical bovine anaplasmosis infections using a subcutaneous ear implant vaccine construct.

Anaplasma ovis genetic diversity detected by major surface protein 1a and its prevalence in small ruminants

Anaplasma ovis is a widely distributed tick-borne rickettsial pathogen of sheep, goats, and wild ruminants. The aims of this study were to assess the prevalence, associations of Anaplasma ovis in sheep and goats, as well as its genetic diversity based on analysis of the msp1α gene. A total of 416 DNA samples from sheep (n = 236) and goats (n = 180) from four provinces in southeastern Turkey were analyzed by PCR. The overall A. ovis prevalence was 18% (CI 14.4–22.1). The infection rates of A. ovis varied from 15.9% to 21.8% in sampled provinces, and they were not significantly different. There was no difference between Anaplasma ovis infection in sheep (20.3%, CI 15.4–26.0) and goats (15.0%, CI 10.1–21.1) or in infection rate of animals <1 year (21.8%, CI 14.9-30.1) compared to >1 year (16.4%, CI 12.4–21.2). A significant association between A. ovis infection and the presence of Rhipicephalus bursa and Rhipicephalus turanicus was observed (P < 0.05). Prevalence of A. ovis-positive animals was higher in animals showing co-infection with Babesia and Theileria compared to those not co-infected (P < 0.05). The Msp1a amino acid repeats were identified and used for the characterization of A. ovis strains. Forty partial msp1a gene sequences containing the repeated sequences of A. ovis were obtained, and 14 previously undescribed tandem repeats with 33 to 43 amino acids were found. Thirteen A. ovis genotypes were identified based on the structure of Msp1a tandem repeats. The majority of A. ovis isolates exhibited one Msp1a tandem repeat, with a maximum of three. This study revealed the Msp1a could be used as a marker for genotyping A. ovis, and high genetic diversity of A. ovis were found in small ruminants in Turkey.

Characterization of Anaplasma ovis strains using the major surface protein 1a repeat sequences

Anaplasma ovis is one of the tick-transmitted pathogens of small ruminants. It causes ovine anaplasmosis and widely distributed in the world. In contrast to extensive worldwide genetic diversity of A. marginale and A. phagocytophilum, there are few reports on the classification of A. ovis strains. This study was conducted to investigate the occurrence and characterize A. ovis strains from goats and sheep from 12 provinces in China. The occurrence of A. ovis DNA was tested in 552 goats and sheep, by PCR based on the msp4 gene. Positive samples were used for the amplification of the msp1a gene of A. ovis. The Msp1a amino acid repeats were further identified and used for the characterization of A. ovis strains. The results showed that 79 (14.3%) goats and sheep were positive for A. ovis. The infection rates of A. ovis among different study sites ranged from 0 to 100%, and were significantly higher in sheep (26.6%, 45/169) than in goats (8.9%, 34/383) (χ 2 = 21.403, df = 1, P < 0.001). The msp4 gene sequences of these isolates were 99.8–100% identical to each other, and they represented two sequence types. Forty-four partial msp1a gene sequences containing the repeat sequences were obtained from A. ovis-positive samples. After translation to amino acid sequences, 24 Msp1a repeats with 33 to 47 amino acids, which corresponded to 19 genotypes of A. ovis, were recognized in goats and sheep in China. Anaplasma ovis is widely distributed in the investigated geographical regions. The msp4 gene of A. ovis had high sequence identity and was unable to be used to discriminate different strains. The Msp1a could be used as a genetic marker for characterizing A. ovis, and 19 genotypes of A. ovis were recognized in domestic small ruminants in China. The present study revealed, for the first time, the genetic diversity of A. ovis based on the analyses of Msp1a amino acid repeats.

Low genetic diversity of Anaplasma marginale in calves in an endemic area for bovine anaplasmosis in the state of São Paulo, Brazil

Major surface protein 1a has been used as a marker for genetic stability in identifying geographical isolates of Anaplasma marginale. We conducted a longitudinal study to ascertain the genetic diversity of A. marginale in a dairy cattle herd with a history of clinical anaplasmosis. A total of 20 calves were evaluated every 3 months, from birth to 1 year of age. They were evaluated using blood smears, IFAT, ELISA, and qPCR. Additionally, samples positive for the msp1a gene using nPCR were sequenced. The detection of A. marginale ranged from 20 to 90% using blood smears, 20–80% using ELISA/IFAT, and 15–100% using qPCR. We found evidence that suggested transplacental transmission of A. marginale in 15% (3/20) of the calves based on qPCR and 20% (4/20) based on blood smears. Additionally, these four animals were A. marginale ELISA/IFAT-positive. The A. marginale strains found were α-β3-Γ (n=7), α-β2 (n=1), and α-β2-190-Γ (n=1). The new MSP1a tandem repeat 190 was described. The results showed that the genetic diversity of A. marginale in a group of calves up to 1 year of age from Taiaçu (SP) was low, with only three different strains identified, showing the microsatellite genotype E. Eighty percent of the animals evaluated had clinical signs of bovine anaplasmosis and were treated using oxytetracycline and imidocarb dipropionate. Additionally, it was found that 30% (6/20) and 10% (2/20) of the animals required a second and third treatment, respectively, based on clinical signs. Four animals infected with the α-β3-Γ strain died, showing that this strain is also involved in the clinical anaplasmosis cases in Brazil.

Genetic diversity and molecular phylogeny of Anaplasma marginale studied longitudinally under natural transmission conditions in Rio de Janeiro, Brazil

Anaplasma marginale is the most prevalent tick-borne pathogen in cattle in tropical and subtropical regions of the world. Major Surface Protein 1a (MSP1a) has been found to be a stable genetic marker for identifying A. marginale isolates within geographical regions. It is conserved in cattle during infection and tick-borne transmission of the pathogen. The aim of the present longitudinal study was to determine occurrences of genetic diversity associated with high prevalence of A. marginale under natural transmission conditions. Twenty calves were evaluated every 3 months during the first year of life. Rickettsemia levels due to A. marginale, measured as the number of msp1αcopies/ml in the blood of positive calves, ranged from 2.06×103 to 4.36×1012. The numbers of MSP1a tandem repeats among MSP1a tandem repeats were 3 and 6. The predominant msp1α microsatellite was E, and another MSP1a tandem repeat was found that presented genotype G. Nineteen different MSP1a tandem repeats of A. marginale were found circulating in animals. The MSP1a tandem repeats 4–63–27 (27.5%), 78–242–25–31 (n=21.6%) and τ–102–15 (n=17.6%) were the ones most commonly observed. Twenty-two MSP1a tandem repeats resulted in new sequences with amino acid changes, as shown in this study. Thirty sequences were found for the first time in Brazil. Glycine, glutamate, serine and alanine amino acids were found at position 20. During the study, 80% (16/20) of the animals were infected by more than one genotype. Three animals were born infected, with MSP1a tandem repeats 4–63–27, 78–242–25–31 and τ–102–15, thus indicating occurrence of transplacental transmission. In the phylogenetic analysis, 19 different MSP1a tandem repeats of A. marginale were found in the cattle, which suggested that many MSP1a tandem repeats and high variation in MSP1a were occurring.

Anaplasma marginale major surface protein 1a: A marker of strain diversity with implications for control of bovine anaplasmosis

Classification of bacteria is challenging due to the lack of a theory-based framework. In addition, the adaptation of bacteria to ecological niches often results in selection of strains with diverse virulence, pathogenicity and transmission characteristics. Bacterial strain diversity presents challenges for taxonomic classification, which in turn impacts the ability to develop accurate diagnostics and effective vaccines. Over the past decade, the worldwide diversity of Anaplasma marginale, an economically important tick-borne pathogen of cattle, has become apparent. The extent of A. marginale strain diversity, formerly underappreciated, has contributed to the challenges of classification which, in turn, likely impacts the design and development of improved vaccines. Notably, the A. marginale surface protein 1a (MSP1a) is a model molecule for these studies because it serves as a marker for strain identity, is both an adhesin necessary for infection of cells and an immuno-reactive protein and is also an indicator of the evolution of strain diversity. Herein, we discuss a molecular taxonomic approach for classification of A. marginale strain diversity. Taxonomic analysis of this important molecule provides the opportunity to understand A. marginale strain diversity as it relates geographic and ecological factors and to the development of effective vaccines for control of bovine anaplasmosis worldwide.

Epidemiology and evolution of the genetic variability of Anaplasma marginale in South Africa

Bovine anaplasmosis caused by infection of cattle with Anaplasma marginale has been considered to be endemic in South Africa, an assumption based primarily on the distribution of the tick vectors of A. marginale and serological studies on the prevalence of anaplasmosis in Limpopo, Free State, and North West. However, molecular evidence of the distribution of anaplasmosis has only been reported in the Free State province. In order to establish effective control measures for anaplasmosis, epidemiological surveys are needed to define the prevalence and distribution of A. marginale in South Africa. In addition, a proposed control strategy for anaplasmosis is the development of an A. marginale major surface protein 1a (MSP1a)-based vaccine. Nevertheless, regional variations of this gene would need to be characterized prior to vaccine development for South Africa. The objectives of the present study were therefore to conduct a national survey of the prevalence of A. marginale in South Africa, followed by an evaluation of the diversity and evolution of msp1a in South African strains of A. marginale. To accomplish these objectives, species-specific PCR was used to test 250 blood samples from cattle collected from all South African provinces (including 26 districts and municipalities), except the Free State province where similar studies were reported previously. The prevalence of A. marginale ranged from 65% to 100%, except in Northern Cape province where A. marginale was not detected. A correlation was found between the prevalence and genetic diversity of A. marginale MSP1a. Additionally, the genetic diversity of the A. marginale MSP1a was found to evolve under negative and positive selection, and 23 new tandem repeats in South Africa were shown to have evolved from the extant tandem repeat 4. Despite the MSP1a genetic variability, some types of tandem repeats were found to be conserved among the A. marginale strains, and low-variable peptides in MSP1a tandem repeats were subsequently identified. The results of this research confirmed that anaplasmosis is endemic in South Africa. The results of the molecular characterization of the MSP1a can then be used as the basis for development of new and novel vaccines for anaplasmosis control in South Africa.

Control of tick infestations and pathogen prevalence in cattle and sheep farms vaccinated with the recombinant Subolesin-Major Surface Protein 1a chimeric antigen

BackgroundDespite the use of chemical acaricides, tick infestations continue to affect animal health and production worldwide. Tick vaccines have been proposed as a cost-effective and environmentally friendly alternative for tick control. Vaccination with the candidate tick protective antigen, Subolesin (SUB), has been shown experimentally to be effective in controlling vector infestations and pathogen infection. Furthermore, Escherichia coli membranes containing the chimeric antigen composed of SUB fused to Anaplasma marginale Major Surface Protein 1a (MSP1a) (SUB-MSP1a) were produced using a simple low-cost process and proved to be effective for the control of cattle tick, Rhipicephalus (Boophilus) microplus and R. annulatus infestations in pen trials. In this research, field trials were conducted to characterize the effect of vaccination with SUB-MSP1a on tick infestations and the prevalence of tick-borne pathogens in a randomized controlled prospective study.MethodsTwo cattle and two sheep farms with similar geographical locations and production characteristics were randomly assigned to control and vaccinated groups. Ticks were collected, counted, weighed and classified and the prevalence of tick-borne pathogens at the DNA and serological levels were followed for one year prior to and 9 months after vaccination.ResultsBoth cattle and sheep developed antibodies against SUB in response to vaccination. The main effect of the vaccine in cattle was the 8-fold reduction in the percent of infested animals while vaccination in sheep reduced tick infestations by 63%. Female tick weight was 32-55% lower in ticks collected from both vaccinated cattle and sheep when compared to controls. The seroprevalence of Babesia bigemina was lower by 30% in vaccinated cattle, suggesting a possible role for the vaccine in decreasing the prevalence of this tick-borne pathogen. The effect of the vaccine in reducing the frequency of one A. marginale msp4 genotype probably reflected the reduction in the prevalence of a tick-transmitted strain as a result of the reduction in the percent of tick-infested cattle.ConclusionsThese data provide evidence of the dual effect of a SUB-based vaccine for controlling tick infestations and pathogen infection/transmission and provide additional support for the use of the SUB-MSP1a vaccine for tick control in cattle and sheep.

Functional and Immunological Relevance of Anaplasma marginale Major Surface Protein 1a Sequence and Structural Analysis

Bovine anaplasmosis is caused by cattle infection with the tick-borne bacterium, Anaplasma marginale. The major surface protein 1a (MSP1a) has been used as a genetic marker for identifying A. marginale strains based on N-terminal tandem repeats and a 5′-UTR microsatellite located in the msp1a gene. The MSP1a tandem repeats contain immune relevant elements and functional domains that bind to bovine erythrocytes and tick cells, thus providing information about the evolution of host-pathogen and vector-pathogen interactions. Here we propose one nomenclature for A. marginale strain classification based on MSP1a. All tandem repeats among A. marginale strains were classified and the amino acid variability/frequency in each position was determined. The sequence variation at immunodominant B cell epitopes was determined and the secondary (2D) structure of the tandem repeats was modeled. A total of 224 different strains of A. marginale were classified, showing 11 genotypes based on the 5′-UTR microsatellite and 193 different tandem repeats with high amino acid variability per position. Our results showed phylogenetic correlation between MSP1a sequence, secondary structure, B-cell epitope composition and tick transmissibility of A. marginale strains. The analysis of MSP1a sequences provides relevant information about the biology of A. marginale to design vaccines with a cross-protective capacity based on MSP1a B-cell epitopes.

Diversidad genética de la región variable de los genes msp1a y msp4 en cepas de Anaplasma marginale de México

Control of anaplasmosis is limited due to the rickettsia's wide genetic diversity. Major surface protein (Msp) 1a is composed of one constant region and a variable region which may contain one or more 23 - 31 aminoacid repeat sequences. Msp1a variable region together with Msp4 have been used for phylogenetic purposes. We analyzed the variable region of Msp1a and Msp4 gene of ten new Mexican A. marginale strains and compared with previously published sequences in order to correlate the structure of these genes with geographic distribution. In the case of Msp1a, there were geographically distant strains that shared the same variable region, whereas strains from the same outbreak or the same region showed very different structures. Wide diversity in this region had been observed with previous analysis yet a pattern composed by several repeats emerged along organisms isolated from the Mexican gulf coast states. This variation can be explained in part by the immune system pressure over the organism, yet other independent transmission events have to be considered. In the case of Msp4, little variation was observed at the nucleotide level which did not affect the amino acid structures along the new strains analyzed, yet there are differences with previously published for Mexican isolates.

Rapid Deletion of Antigen-Specific CD4+ T Cells following Infection Represents a Strategy of Immune Evasion and Persistence for Anaplasma marginale

Acquired T cell immunity is central for protection against infection. However, the immunological consequences of exposing memory T cells to high Ag loads during acute and persistent infection with systemic pathogens are poorly understood. We investigated this by using infection with Anaplasma marginale, a ruminant pathogen that replicates to levels of 10(9) bacteria per ml of blood during acute infection and maintains mean bacteremia levels of 10(6) per ml during long-term persistent infection. We established that immunization-induced Ag-specific peripheral blood CD4(+) T cell responses were rapidly and permanently lost following infection. To determine whether these T cells were anergic, sequestered in the spleen, or physically deleted from peripheral blood, CD4(+) T lymphocytes from the peripheral blood specific for the major surface protein (MSP) 1a T cell epitope were enumerated by DRB3*1101 tetramer staining and FACS analysis throughout the course of immunization and challenge. Immunization induced significant epitope-specific T lymphocyte responses that rapidly declined near peak bacteremia to background levels. Concomitantly, the mean frequency of tetramer(+)CD4(+) cells decreased rapidly from 0.025% before challenge to a preimmunization level of 0.0003% of CD4(+) T cells. Low frequencies of tetramer(+)CD4(+) T cells in spleen, liver, and inguinal lymph nodes sampled 9-12 wk postchallenge were consistent with undetectable or unsustainable Ag-specific responses and the lack of T cell sequestration. Thus, infection of cattle with A. marginale leads to the rapid loss of Ag-specific T cells and immunologic memory, which may be a strategy for this pathogen to modulate the immune response and persist.

Genetic diversity of major surface protein 1a of Anaplasma marginale in beef cattle

The present study was aimed to demonstrate genotypic diversity of Anaplama marginale in infected beef herds grazing within anaplasmosis endemic regions. The genotypic diversity was identified among different herds, within each herd, and also within single animals. The Israeli strains revealed unique characteristics of MSP1a repeats and, in addition to the published repeats, six new tandem repeats designated Is1–5, and Is9 were identified. The superinfections of individual Anaplama centrale vaccinated animals with two genotypically different A. marginale strains were detected. Six out of 43 vaccinated animals in the G herd were each infected with two A. marginale strains carrying two distinct genotypes; in this herd the follow-up during years 2003–2007 demonstrated that several animals carried different msp1a genotypes at different time points. Coinfection with two different genotypes of A. marginale in A. centrale vaccinated cattle was observed in another herd, as well. It appears that A. marginale is composed of a heterogeneous changing bacterial population that evolves in the host or, the genotypic diversity implies high transmission intensity by the vector, or both. Learning how this diversity is generated and identification of distinct A. marginale strains coupled with high sequence variation of MSP1a will aid in understanding Anaplasma transmission and disease development.

Anaplasma marginale major surface protein 1a directs cell surface display of tick BM95 immunogenic peptides on Escherichia coli

The surface display of heterologous proteins on live Escherichia coli using anchoring motifs from outer membranes proteins has impacted on many areas of biochemistry, molecular biology and biotechnology. The Anaplasma marginale major surface protein 1a (MSP1a) contains N-terminal surface-exposed repeated peptides (28–289 amino acids) that are involved in pathogen interaction with host cell receptors and is surface-displayed when the recombinant protein is expressed in E. coli. Therefore, it was predicted that MSP1a would surface display on E. coli peptides inserted in the N-terminal repeats region of the protein. The Rhipicephalus ( Boophilus) microplus BM86 and BM95 glycoproteins are homologous proteins that protect cattle against tick infestations. In this study, we demonstrated that a recombinant protein comprising tick BM95 immunogenic peptides fused to the A. marginale MSP1a N-terminal region is displayed on the E. coli surface and is recognized by anti-BM86 and anti-MSP1a antibodies. This system provides a novel approach to the surface display of heterologous antigenic proteins on live E. coli and suggests the possibility to use the recombinant bacteria for immunization studies against cattle tick infestations.

Physical Linkage of Naturally Complexed Bacterial Outer Membrane Proteins Enhances Immunogenicity

The outer membrane proteins (OMPs) of bacterial pathogens are essential for their growth and survival and especially for attachment and invasion of host cells. Since the outer membrane is the interface between the bacterium and the host cell, outer membranes and individual OMPs are targeted for development of vaccines against many bacterial diseases. Whole outer membrane fractions often protect against disease, and this protection cannot be fully reproduced by using individual OMPs. Exactly how the interactions among individual OMPs influence immunity is not well understood. We hypothesized that one OMP rich in T-cell epitopes can act as a carrier for an associated OMP which is poor in T-cell epitopes to generate T-dependent antibody responses, similar to the hapten-carrier effect. Major surface protein 1a (MSP1a) and MSP1b1 occur as naturally complexed OMPs in the Anaplasma marginale outer membrane. Previous studies demonstrated that immunization with the native MSP1 heteromer induced strong immunoglobulin G (IgG) responses to both proteins, but only MSP1a stimulated strong CD4+ T-cell responses. Therefore, to test our hypothesis, constructs of CD4+ T-cell epitopes from MSP1a linked to MSP1b1 were compared with individually administered MSP1a and MSP1b1 for induction of MSP1b-specific IgG. By linking the T-cell epitopes from MSP1a to MSP1b1, significantly higher IgG titers against MSP1b1 were induced. Understanding how the naturally occurring intermolecular interactions between OMPs influence the immune response may lead to more effective vaccine design.

Phylogenetic Analysis of Anaplasma marginale Strains from Paraná State, Brazil, Using the msp1α and msp4 genes

Anaplasma marginale is an obligate intraerythrocytic rickettsial pathogen (order, Rickettsiales: family, Anaplasmataceae) that causes bovine anaplasmosis. This disease is widely distributed in tropical and sub-tropical regions of the world and causes important economic losses to cattle production. Major surface protein (MSP)1a (msp1alpha gene) is one of the six MSPs identified on A. marginale from cattle, whose sequence and size vary according to the number of tandem 28- to 29-amino acid repeats. This study characterized the msp1alpha and msp4 genes obtained from three distinct Brazilian herds from the State of Paraná. Three strains of the msp1alpha and one strain of the msp4 gene were sequenced. The strains evaluated revealed PCR products of different size, representing three, five and six internal repeats. Sequence analyses confirmed the number of tandem sequence copies and revealed a high degree of sequence identity with strains from other Brazilian States, as well as strains from the USA, Europe and Israel. The msp1alpha DNA and amino acid sequences from A. marginale and DNA sequences of msp4 strains did not reveal distinct phylogeographical segregation. However, the amino acid sequences of msp4 demonstrated definite phylogeographical relationship. These results suggest that the amino acid sequences of msp4 should be used for phylogenetic identification of A. marginale strains and may be an important tool for the epidemiology and control of anaplasmosis. Additionally, the close similarity of the Paraná strains of A. marginale with strains from USA, Europe and Asia may reflect the introduction of these genes during the development of the Brazilian bovine herd.

Recent Studies on the Characterization of Anaplasma marginale Isolated from North American Bison

Anaplasma marginale (Rickettsiales: Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world. Many geographic isolates of A. marginale occur worldwide that have been identified by major surface protein (MSP) 1a, which varies in sequence and molecular weight owing to different numbers of tandem 28-29 amino acid repeats. Although serologic tests have identified American bison, Bison bison, as being infected with A. marginale, the present studies were undertaken to confirm A. marginale infection in bison, to characterize bison isolates, and to compare the phylogenetic relationship of the bison isolates with other A. marginale isolates from North America. Nine A. marginale isolates derived from Canadian bison possessed identical msp4 sequences with one characteristic silent nucleotide change. The sequence of MSP1a was determined for one Canadian and two U.S. bison isolates of A. marginale, and these isolates contained 4 and 5 tandem repeats, respectively. One U.S. bison isolate tested for infectivity proved to be infective for cattle and transmitted by Dermacentor variabilis ticks. the results of this study demonstrated that these A. marginale isolates obtained from bison were similar to ones derived from naturally infected cattle.

Glycosylation of Anaplasma marginale major surface protein 1a and its putative role in adhesion to tick cells.

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.