Expression and immune recognition of the conserved MSP4 outer membrane protein of Anaplasma marginale.
ABSTRACT: Defining conserved, protective epitopes is essential to the design of an effective vaccine against bovine anaplasmosis. MSP4, one of six initial body proteins recognized by a neutralizing serum, is conserved among Anaplasma marginale isolates at both the protein and the DNA levels. Sera from cattle immunized with an outer membrane fraction of A. marginale and protected from a virulent challenge bind MSP4. The gene for MSP4 has been cloned, and the recombinant protein has been expressed, isolated, and demonstrated to share epitopes with the native protein expressed on initial bodies. MSP4 may have a greater potential to protect cattle from a challenge by heterologous isolates than other A. marginale surface proteins, which vary widely in size and structure.
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.
Project description:Anaplasma marginale (order Rickettsiales, family Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world. Many geographic isolates of A. marginale occur in the United States and have been identified by major surface protein 1a (MSP1a), which varies in sequence and molecular weight due to different numbers of tandem 28- to 29-amino-acid repeats. The present study was undertaken to examine the genetic variations among isolates of A. marginale obtained during 2001 from infected cattle from east-central Oklahoma, where A. marginale is endemic. The gene and protein sequences of MSP1a and msp4 nucleotide sequences were used to infer the phylogenetic relationships among Oklahoma and New World isolates from Argentina, Brazil, Mexico, and the United States. All 11 A. marginale isolates collected from Oklahoma had different MSP1a sequences but identical MSP4 sequences. The phylogenies of the msp4 sequences of 13 isolates from Oklahoma in comparison with those of 7 Latin American isolates and 12 U.S. isolates by maximum-parsimony (MP) and maximum-likelihood (ML) analyses, with A. centrale and A. ovis sequences used as outgroups, provided strong bootstrap analysis support for a Latin American clade. Isolates of A. marginale from the southern United States (Florida, Mississippi, and Virginia) and the west-central United States (California, Idaho, Illinois, Oregon, Missouri, and Texas) also grouped into two clades. Both clades contained isolates from Oklahoma, suggesting extensive cattle movement. ML analysis of the msp4 sequences of isolates from Oklahoma provided bootstrap analysis support for east-central and north-central clades in Oklahoma, and both clades included isolates from Stillwater, Okla. Analysis of the codon and amino acid changes among the msp4 sequences of isolates with different phylogenies provided evidence that msp4 is not under positive selection pressure. In contrast, the phylogenies of the MSP1a DNA and protein sequences of 13 isolates from Oklahoma in comparison with those of 7 Latin American and 13 isolates from the United States by MP and ML analyses demonstrated no geographic clustering and provided evidence that this gene is under positive selection pressure. The results indicate that msp1alpha is not a marker for the characterization of A. marginale geographic isolates and suggest that the genetic heterogeneity observed among isolates of A. marginale within Oklahoma could be explained by cattle movement and the maintenance of different genotypes by independent transmission events.
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.
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.
Project description:Anaplasma marginale is an important tick-transmitted rickettsial pathogen of cattle, with worldwide distribution and an important economic impact. The genetic diversity of A. marginale strains has been extensively characterized in different geographical regions throughout the world, while information is limited on studies in China. This study was carried out to determine the prevalence and genetic diversity of A. marginale strains in cattle from ten provinces of China.A total of 557 blood samples from cattle were collected and screened for the occurrence of A. marginale by PCR based on the msp4 gene. The partial msp1a gene containing tandem repeat sequences was further amplified from msp4 positive samples. The Msp1a amino acid repeats were identified, and genetic variation of A. marginale strains was characterized based on the variation in the repeated portion of Msp1a.Our results showed that 31.6% of 557 cattle were positive for A. marginale. The infection rates of A. marginale varied considerably from 0 to 96.9% in different sampling regions. Sequence analysis revealed that two msp4 sequence variants of A. marginale exist in cattle. One hundred and three msp1a sequences were obtained and permitted to identify 42 Msp1a tandem repeats, 21 of which were not previously published for A. marginale. Moreover, 61 A. marginale genotypes were identified based on the structure of Msp1a tandem repeats.Anaplasma marginale is widely distributed in China and a high prevalence of infection was observed in cattle. The geographical strains of A. marginale were molecularly characterized based on the structure of Msp1a tandem repeats. Forty-two Msp1a tandem repeats and 61 genotypes of A. marginale were identified. This study, for the first time, revealed the genetic diversity of A. marginale strains in cattle in China.
Project description:Anaplasmosis is one of several tick-borne diseases severely constraining cattle production and usage in many parts of the world. Cattle can be protected from anaplasmosis by immunization with major surface protein 1, a surface protein of Anaplasma marginale carrying a neutralization-sensitive epitope. Marked size polymorphisms exist among different isolates of A. marginale in the AmF105 subunit of major surface protein 1, yet all isolates still contain the neutralization-sensitive epitope. To clarify the basis for these observations, the mspl alpha gene encoding AmF105 was cloned from four isolates and sequenced. The encoded polypeptides share a high degree of overall homology between isolates but contain a domain with various numbers of tandemly repeated sequences and three regions of clustered amino acid substitutions outside the repeat domain. The polypeptide size differences are completely explained by the variations in the numbers of tandem repeat units. We have mapped the neutralization-sensitive epitope to a sequence that is present within each repeat unit. These results identify a basis for size polymorphisms of the surface polypeptide antigen concomitant with B-cell epitope conservation in rickettsiae.
Project description:Tick-borne diseases caused by Anaplasma species put serious constraints on the health and production of domestic cattle in tropical and sub-tropical regions. After recovering from a primary infection, cattle typically become persistent carriers of pathogens and play a critical role in the epidemiology of the disease, acting as reservoirs of the Anaplasma spp.In this study a duplex PCR assay was used for the simultaneous detection of Anaplasma marginale and Anaplasma phagocytophilum in cattle using two primer pairs targeting msp4 and msp2 genes, respectively. We used this method to analyze DNA preparations derived from 328 blood cattle samples that were collected from 80 farms distributed among Tunisia's four bioclimatic zones.The prevalence of the A. marginale infection (24.7 %) was significantly higher and more widespread (in all bioclimatic areas) than that of A. phagocytophilum (0.6 %), which was found in a mixed infection with A. marginale.The duplex PCR assay used proved to be a rapid, specific and inexpensive mean for the simultaneous detection of Anaplasma marginale and Anaplasma phagocytophilum in cattle blood. It allowed us to report the identification of A. phagocytophilum for the first time in cattle in Tunisia and confirm the presence of A. marginale in cattle from several geographical areas of the country. Further epidemiological studies undertaken using this assay will help improve the surveillance of the associated diseases in the regions where they are endemic.
Project description: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.
Project description:Anaplasma marginale is the most prevalent tick-borne livestock pathogen with worldwide distribution. Bovine anaplasmosis is a significant threat to cattle industry. Anaplasmosis outbreaks in endemic areas are prevented via vaccination with live A. centrale produced in splenectomized calves. Since A. centrale live vaccine can carry other pathogens and cause disease in adult cattle, research efforts are directed to develop safe recombinant subunit vaccines. Previous work found that the subdominant proteins of A. marginale type IV secretion system (T4SS) and the subdominant elongation factor-Tu (Ef-Tu) were involved in the protective immunity against the experimental challenge in cattle immunized with the A. marginale outer membrane (OM). This study evaluated the immunogenicity and protection conferred by recombinant VirB9.1, VirB9.2, VirB10, VirB11, and Ef-Tu proteins cloned and expressed in E. coli. Twenty steers were randomly clustered into four groups (G) of five animals each. Cattle from G1 and G2 were immunized with a mixture of 50 ?g of each recombinant protein with Quil A® or Montanide™ adjuvants, respectively. Cattle from G3 and G4 (controls) were immunized with Quil A and Montanide adjuvants, respectively. Cattle received four immunizations at three-week intervals and were challenged with 107 A. marginale-parasitized erythrocytes 42 days after the fourth immunization. After challenge, all cattle showed clinical signs, with a significant drop of packed cell volume and a significant increase of parasitized erythrocytes (p<0.05), requiring treatment with oxytetracycline to prevent death. The levels of IgG2 induced in the immunized groups did not correlate with the observed lack of protection. Additional strategies are required to evaluate the role of these proteins and their potential utility in the development of effective vaccines.
Project description:Bovine anaplasmosis caused by Anaplasma marginale represents a serious threat to cattle farming worldwide, especially in the tropics and subtropics. In the present study, archived DNA samples from the blood of cattle (n=437) in the Nuwara Eliya, Galle, Ampara, Polonnaruwa, and Jaffna districts and buffalo (n=327) in the Galle, Polonnaruwa, Mannar, and Mullaitivu districts in Sri Lanka, were screened for A. marginale using a major surface protein 5 (msp5) gene-based PCR assay. The findings showed that 32.7 and 57.5% of cattle and buffalo, respectively, were A. marginale-positive. The rate of positivity differed significantly among geographical regions. In conclusion, the high rates of A. marginale infection in cattle and buffalo highlight the importance of effective control measures in Sri Lanka.