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.

scholarly journals Major Surface Protein 2 of Anaplasma phagocytophilum Facilitates Adherence to Granulocytes

2003 ◽  
Vol 71 (7) ◽  
pp. 4018-4025 ◽  
Author(s):  
Jinho Park ◽  
Kyoung Seong Choi ◽  
J. Stephen Dumler
Keyword(s):  
Monoclonal Antibody ◽  
Bacterial Adhesion ◽  
Anaplasma Phagocytophilum ◽  
Human Peripheral Blood ◽  
Myeloid Cells ◽  
Surface Protein ◽  
Mammalian Host ◽  
Blood Neutrophils ◽  
Major Surface

ABSTRACT Anaplasma phagocytophilum is an obligate intracellular bacterium that infects myeloid cells in the mammalian host. Msp2 (p44) is the major immunodominant outer-membrane protein of these bacteria. We hypothesized that Msp2 acts as an adhesin for A. phagocytophilum entry into granulocytes. This potential role was investigated by blocking binding with Msp2 monoclonal antibodies and by antagonizing binding and propagation with recombinant Msp2 (rMsp2) in vitro. With HL-60 cells, fresh human peripheral blood neutrophils, and a cell line devoid of the fucosylated platelet selectin glycoprotein ligand 1 (PSGL-1) receptor for A. phagocytophilum or one that was transfected to express this ligand, Msp2 monoclonal antibody and rMsp2 used as the antagonist caused concentration-dependent reductions in bacterial adhesion (P < 0.007 and P < 0.02, respectively) and propagation (P < 0.05 and P < 0.001), although inhibition of adhesion or propagation was moderate and incomplete. Likewise, rMsp2 bound to surfaces of the transfected cell at a level similar to that of extracellular A. phagocytophilum and significantly (P < 0.05) beyond that of nontransfected cells. Moreover, a dose-dependent reduction (P < 0.019) in PSGL-1 monoclonal antibody binding to HL-60 cells was elicited with rMsp2. We conclude that Msp2s of A. phagocytophilum are involved in bacterial adhesion to ligands on host myeloid cells before intracellular infection.

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 Characterization of Anaplasma marginale Isolated from North American Bison

2003 ◽  
Vol 69 (8) ◽  
pp. 5001-5005 ◽  
Author(s):  
José de la Fuente ◽  
Elizabeth J. Golsteyn Thomas ◽  
Ronald A. Van Den Bussche ◽  
Robert G. Hamilton ◽  
Elaine E. Tanaka ◽  
...  
Keyword(s):  
New World ◽  
Surface Protein ◽  
Anaplasma Marginale ◽  
The United States ◽  
Bison Bison ◽  
Infected Cattle ◽  
Major Surface Protein ◽  
The World ◽  
Major Surface

ABSTRACT 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.

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 Serotyping Isolates of Anaplasma phagocytophilum by Using Monoclonal Antibodies

2003 ◽  
Vol 10 (5) ◽  
pp. 969-972 ◽  
Author(s):  
Hisashi Inokuma ◽  
Philippe Brouqui ◽  
J. Stephen Dumler ◽  
Didier Raoult
Keyword(s):  
New York ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Blot Analysis ◽  
Anaplasma Phagocytophilum ◽  
Granulocytic Ehrlichiosis ◽  
Human Granulocytic Ehrlichiosis ◽  
Antigenic Characterization ◽  
Animal Isolates

ABSTRACT Ten mouse monoclonal antibodies (MAbs) that react with Anaplasma phagocytophilum (the human granulocytic ehrlichiosis agent) Webster isolates were developed. Seven different isolates of A. phagocytophilum were subtyped with these MAbs. Western blot analysis revealed that these MAbs reacted mainly with 41- to 46-kDa Msp2 proteins. Six MAbs reacted with all isolates. Four other MAbs reacted with human isolates from Wisconsin, but not with human isolates from New York or with animal isolates. Three different serotypes were identified. These features may lead to the development of other specific MAbs in order to provide tools for antigenic characterization of human isolates of A. phagocytophilum.

Expression and antigenic characterization of recombinant Mycoplasma agalactiae P48 major surface protein

2000 ◽  
Vol 71 (3-4) ◽  
pp. 201-210 ◽  
Author(s):  
S. Rosati ◽  
P. Robino ◽  
M. Fadda ◽  
S. Pozzi ◽  
A. Mannelli ◽  
...  
Keyword(s):  
Surface Protein ◽  
Mycoplasma Agalactiae ◽  
Major Surface Protein ◽  
Antigenic Characterization ◽  
Major Surface
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