scholarly journals Expression and Immunogenicity of Recombinant Immunoreactive Surface Protein 2 of Anaplasma phagocytophilum

2012 ◽  
Vol 19 (6) ◽  
pp. 919-923 ◽  
Author(s):  
Qiang Yu ◽  
Chuang-fu Chen ◽  
Qiang Chen ◽  
Li-juan Zhang
Keyword(s):  
Recombinant Protein ◽  
Anaplasma Phagocytophilum ◽  
Surface Protein ◽  
Immunofluorescence Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type ◽  
Major Surface Protein ◽  
Granulocytic Anaplasmosis ◽  
Major Surface ◽  
Diagnostic Reagent

ABSTRACTHuman granulocytic anaplasmosis (HGA), caused byAnaplasma phagocytophilum, is an emerging tick-borne zoonotic disease throughout the world. The first HGA cases in China were documented in 2008, and the greatest challenge posed by the disease is rapid and accurate diagnosis during the acute phage of illness. In this study, we successfully cloned and expressed anA. phagocytophilumimmunoreactive surface protein (major surface protein 2 [MSP2]) and demonstrated that this recombinant protein has natural immunogenicity by Western blotting and enzyme-linked immunosorbent assay (ELISA) using human HGA-positive sera and reference rabbit HGA-positive sera. The rabbit antisera against the recombinant protein also reacted actively with the natural antigen ofA. phagocytophilumby immunofluorescence assay (IFA). No cross-reaction was observed between the recombinant protein and rabbit antisera against 10 common members of the orderRickettsialesby ELISA when the sera were diluted more than 1:100. We concluded that the recombinant MSP2 protein exhibited excellent antigenicity and specificity, results that should lay the foundation for the development of a simple and rapid diagnostic reagent and a vaccination for anaplasmosis.

scholarly journals Characterization of Anaplasma phagocytophilum Major Surface Protein 5 and the Extent of Its Cross-Reactivity with A. marginale

2007 ◽  
Vol 14 (3) ◽  
pp. 262-268 ◽  
Author(s):  
N. I. Strik ◽  
A. R. Alleman ◽  
A. F. Barbet ◽  
H. L. Sorenson ◽  
H. L. Wamsley ◽  
...  
Keyword(s):  
United States ◽  
Anaplasma Phagocytophilum ◽  
Indirect Elisa ◽  
Surface Protein ◽  
Anaplasma Marginale ◽  
The United States ◽  
Cross Reactivity ◽  
Serum Samples ◽  
Major Surface Protein ◽  
Major Surface

ABSTRACT Major surface protein 5 (Msp5) of Anaplasma marginale is highly conserved in the genus Anaplasma and the antigen used in a commercially available competitive enzyme-linked immunosorbent assay (cELISA) for serologic identification of cattle with anaplasmosis. This study analyzes the degrees of conservation of Msp5 among various isolates of Anaplasma phagocytophilum and the extent of serologic cross-reactivity between recombinant Msp5 (rMsp5) of Anaplasma marginale and A. phagocytophilum. The msp5 genes from various isolates of A. phagocytophilum were sequenced and compared. rMsp5 proteins of A. phagocytophilum and A. marginale were used separately in an indirect ELISA to detect cross-reactivity in serum samples from humans and dogs infected with A. phagocytophilum and cattle infected with A. marginale. Serum samples were also tested with a commercially available competitive ELISA that uses monoclonal antibody ANAF16C1. There were 100% sequence identities in the msp5 genes among all of the A. phagocytophilum isolates from the United States and a horse isolate from Sweden. Sheep isolates from Norway and dog isolates from Sweden were 99% identical to one another but differed in 17 base pairs from the United States isolates and the horse isolate. Serologic cross-reactivity was identified when serum samples from cattle infected with A. marginale were reacted with rMsp5 of A. phagocytophilum and when serum samples from humans and dogs infected with A. phagocytophilum were reacted with rMsp5 of A. marginale in an indirect-ELISA format. Serum samples from dogs or humans infected with A. phagocytophilum did not cross-react with rMsp5 of A. marginale when tested with the commercially available cELISA. These results suggest that rMsp5 of A. phagocytophilum is highly conserved among United States and European isolates and that serologic distinction between A. phagocytophilum and A. marginale infections cannot be accomplished if rMsp5 from either organism is used in an indirect ELISA.

scholarly journals Detection of Cattle Naturally Infected with Anaplasma marginale in a Region of Endemicity by Nested PCR and a Competitive Enzyme-Linked Immunosorbent Assay Using Recombinant Major Surface Protein 5

1998 ◽  
Vol 36 (3) ◽  
pp. 777-782 ◽  
Author(s):  
Susana Torioni de Echaide ◽  
Donald P. Knowles ◽  
Travis C. McGuire ◽  
Guy H. Palmer ◽  
Carlos E. Suarez ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Immunosorbent Assay ◽  
Nested Pcr ◽  
Surface Protein ◽  
Anaplasma Marginale ◽  
Epidemiological Studies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Major Surface Protein ◽  
Persistent Infections ◽  
Major Surface

A competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5 (rMSP5-cELISA) of Anaplasma marginale was validated in a naturally infected cattle herd in an area of eastern Oregon where A. marginale is endemic. The true positive and negative A. marginale infection status of 235 randomly selected cattle was determined by using a nested PCR (nPCR) coupled with msp5 sequence analysis and hybridization. Judgment of the reliability of the nPCR and hybridization for detection of persistent infections was based on three observations. First, the nPCR was able to detect as few as 30 infected erythrocytes per ml. Second, the nPCR was able to consistently detect low levels of rickettsemia in seven carrier cattle experimentally infected with A. marginale. Third, msp5sequence analysis showed >95% identity among 30 nPCR amplicons from cattle naturally infected with field strains of A. marginale. The nPCR and hybridization identified 151 infected and 84 uninfected cattle among the 235 animals tested. With a cutoff point of 28%, the rMSP5-cELISA showed a sensitivity of 96% and a specificity of 95%. These results indicate that the rMSP5-cELISA can sensitively and specifically detect cattle with naturally acquired persistent A. marginale infections and suggest that it is an excellent assay for epidemiological studies, eradication programs, and regulation of international cattle movement.

scholarly journals Sensitive and Specific Identification of Neospora caninum Infection of Cattle Based on Detection of Serum Antibodies to Recombinant Ncp29

2002 ◽  
Vol 9 (3) ◽  
pp. 611-615 ◽  
Author(s):  
Daniel K. Howe ◽  
Keliang Tang ◽  
Patricia A. Conrad ◽  
Karen Sverlow ◽  
J. P. Dubey ◽  
...  
Keyword(s):  
Neospora Caninum ◽  
Surface Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sensitive Assay ◽  
Serum Antibodies ◽  
Caninum Infection ◽  
Major Surface Protein ◽  
Major Surface ◽  
Negative Controls ◽  
Important Disease

ABSTRACT Neosporosis is an economically important disease of dairy cattle caused by the protozoan Neospora caninum. Diagnostic tests for neosporosis are complicated by the potential for cross-reaction of antibodies to antigens that are similar between N. caninum and closely related parasites Toxoplasma gondii and Sarcocystis cruzi. To provide a sensitive and specific assay for detecting antibodies to N. caninum in the serum of infected animals, we have investigated a recombinant form of the antigen known as Ncp29 (rNcp29), which is a major surface protein of the parasite. Ncp29 is encoded by a gene that is homologous to the SAG1 gene previously characterized from T. gondii. An enzyme-linked immunosorbent assay (ELISA) was used to screen animals for the presence of serum antibodies specific to rNcp29. The rNcp29 ELISA readily distinguished between cattle known to be infected with N. caninum (optical density [OD] > 1.2 at 1:500 or greater dilution) and negative controls (OD < 0.5 at 1:500). Additionally, sera from animals that were infected with T. gondii or S. cruzi were negative. The rNcp29 ELISA developed here provides a specific and sensitive assay for detecting neosporosis in cattle.

scholarly journals Serologic Cross-Reactivity between Anaplasma marginale and Anaplasma phagocytophilum

2005 ◽  
Vol 12 (10) ◽  
pp. 1177-1183 ◽  
Author(s):  
U. M. Dreher ◽  
J. de la Fuente ◽  
R. Hofmann-Lehmann ◽  
M. L. Meli ◽  
N. Pusterla ◽  
...  
Keyword(s):  
Animal Species ◽  
Surface Protein ◽  
Anaplasma Marginale ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Assay ◽  
Serological Tests ◽  
Major Surface Protein ◽  
New Findings ◽  
Major Surface

ABSTRACT In the context of a serosurvey conducted on the Anaplasma marginale prevalence in Swiss cattle, we suspected that a serological cross-reactivity between A. marginale and A. phagocytophilum might exist. In the present study we demonstrate that cattle, sheep and horses experimentally infected with A. phagocytophilum not only develop antibodies to A. phagocytophilum (detected by immunofluorescent-antibody assay) but also to A. marginale (detected by a competitive enzyme-linked immunosorbent assay). Conversely, calves experimentally infected with A. marginale also developed antibodies to A. phagocytophilum using the same serological tests. The identity of 63% determined in silico within a 209-amino-acid sequence of major surface protein 5 of an isolate of A. marginale and one of A. phagocytophilum supported the observed immunological cross-reactivity. These observations have important consequences for the serotesting of both, A. marginale and A. phagocytophilum infection of several animal species. In view of these new findings, tests that have been considered specific for either infection must be interpreted carefully.

scholarly journals Differential Expression and Glycosylation of Anaplasma phagocytophilum Major Surface Protein 2 Paralogs during Cultivation in Sialyl Lewis x-Deficient Host Cells

2009 ◽  
Vol 77 (5) ◽  
pp. 1746-1756 ◽  
Author(s):  
Matthew J. Troese ◽  
Madhubanti Sarkar ◽  
Nathan L. Galloway ◽  
Rachael J. Thomas ◽  
Sarah A. Kearns ◽  
...  
Keyword(s):  
Differential Expression ◽  
Cell Lines ◽  
Anaplasma Phagocytophilum ◽  
Surface Protein ◽  
Host Cells ◽  
Sialyl Lewis X ◽  
Bacterial Populations ◽  
Major Surface Protein ◽  
Sialyl Lewis ◽  
Major Surface

ABSTRACT Many microbial pathogens alter expression and/or posttranslational modifications of their surface proteins in response to dynamics within their host microenvironments to retain optimal interactions with their host cells and/or to evade the humoral immune response. Anaplasma phagocytophilum is an intragranulocytic bacterium that utilizes sialyl Lewis x (sLex)-modified P-selectin glycoprotein ligand 1 as a receptor for infecting myeloid cells. Bacterial populations that do not rely on this receptor can be obtained through cultivation in sLex-defective cell lines. A. phagocytophilum major surface protein 2 [Msp2(P44)] is encoded by members of a paralogous gene family and is speculated to play roles in host adaptation. We assessed the complement of Msp2(P44) paralogs expressed by A. phagocytophilum during infection of sLex-competent HL-60 cells and two HL-60 cell lines defective for sLex expression. Multiple Msp2(P44) and N-terminally truncated 25- to 27-kDa isoforms having various isoelectric points and electrophoretic mobilities were expressed in each cell line. The complement of expressed msp2(p44) paralogs and the glycosyl residues modifying Msp2(P44) varied considerably among bacterial populations recovered from sLex-competent and -deficient host cells. Thus, loss of host cell sLex expression coincided with both differential expression and glycosylation of A. phagocytophilum Msp2(P44). This reinforces the hypothesis that this bacterium is able to generate a large variety of surface-exposed molecules that could provide great antigenic diversity and result in multiple binding properties.

scholarly journals CHARACTERIZATION OF HUMAN GRANULOCYTIC EHRLICHIOSIS IN EXPERIMENTAL INFECTED HL-60 CELLS WITH ANAPLASMA PHAGOCYTOPHILUM USING NESTED PCR AND A. PHAGOCYTOPHILUM MAJOR SURFACE PROTEIN-2 MONOCLONAL ANTIBODY

2012 ◽  
Vol 3 (2) ◽  
pp. 160-165
Author(s):  
MS Rahman ◽  
JH Park ◽  
JS Chae
Keyword(s):  
Monoclonal Antibody ◽  
Nested Pcr ◽  
Anaplasma Phagocytophilum ◽  
Surface Protein ◽  
Intense Band ◽  
Major Surface Protein ◽  
Granulocytic Ehrlichiosis ◽  
Human Granulocytic Ehrlichiosis ◽  
Major Surface

The study was carried out to characterize the human granulocytic ehrlichiosis in experimental infected HL-60 cells with Anaplasma phagocytophilum using nested PCR and A. phagocytophilum major surface protein-2 monoclonal antibody. The nested PCR revealed only one band of 926 base pair DNA from A. phagocytophilum infected HL-60 cells. The western blot revealed several bands with a dominant 44-kDa. There were intense band of 100- and 160-kDa bands. The 44-kDa component was at least 10 times more abundant than the 100- and 160-kDa bands. In conclusion, the nested PCR would be a valuable tool for the characterization of human granulocytic ehrlichiosis.

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