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

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.The order Rickettsiales represents obligate intracellular bacteria that reside in vacuoles of eukaryotic cells, with the potential to cause fatal tick-transmitted diseases in humans and several mammalian species. Recent genetic studies reorganized some species within the order Rickettsiales, between the families Rickettsiaceae and Anaplasmataceae (11). Based on these studies, three organisms, formerly known as Ehrlichia phagocytophila, Ehrlichia equi, and the HGE (human granulocytic ehrlichiosis) agent, were unified as a single species and moved to the genus Anaplasma. These three organisms are now reclassified as Anaplasma phagocytophilum, the causative agent of granulocytic anaplasmosis, an emerging tick-borne disease (6, 11).A. phagocytophilum has been detected worldwide, particularly in North America and Europe as well as in South Africa, South America, and Asia; it infects humans, horses, ruminants, cats, dogs, and a variety of wildlife species, including rodents, deer, and carnivores (4,

Detecção de anticorpos para Anaplasma sp. em pequenos ruminantes no semi-árido do estado de Pernambuco, Brasil

“…têm possibilitado o desenvolvimento de ensaios de imunoadsorção enzimática (ELISA), baseados em proteínas recombinantes de A. marginale (ARAÚJO et al, 2005), dentre as quais MSP5 (SILVA et al, 2006). Essas pesquisas têm oferecido também uma grande gama de informações sobre a conservação dessas proteínas no gênero (DREHER et al, 2005;STRIK et al, 2007), inclusive com reações sorológicas cruzadas descritas entre MSP5 de A. marginale, A. centrale, A. phagocytophilum e A. ovis (DREHER et al, 2005), e entre MSP5 de A. phagocytophilum e A. platys, Ehrlichia canis e E. chaffeensis (STRIK et al, 2007).…”

“…ou Ehrlichia sp., principalmente se utilizados para animais passíveis de infecção por mais de uma espécie (DREHER et al, 2005). No entanto, representam importantes ferramentas de triagem para detecção das infecções por Anaplasma/Ehrlichia em diversos hospedeiros (STRIK et al, 2007).…”

Serological cross-reactivity between Anaplasma marginale and an Ehrlichia species in naturally and experimentally infected cattle

“…6 In another study, cross-reactivity was not demonstrated between human or dog antibodies to A. phagocytophilum and the recombinant MSP5 antigen of A. marginale when tested by cELISA. 25 However, cross-reactivity was evident when recombinant MSP5 antigens of A. phagocytophilum or A. marginale were incubated with heterologous sera using an indirect ELISA format. 25 A newly described Ehrlichia genotype (BOV2010) was recently detected in naturally infected cattle from British Columbia, Canada.…”

“…25 However, cross-reactivity was evident when recombinant MSP5 antigens of A. phagocytophilum or A. marginale were incubated with heterologous sera using an indirect ELISA format. 25 A newly described Ehrlichia genotype (BOV2010) was recently detected in naturally infected cattle from British Columbia, Canada. 8 Based on the results of cELISA, a the cattle were originally suspected of being infected with A. marginale.…”

Real time polymerase chain reaction to diagnose Anaplasma marginale in cattle and deer (Ozotoceros bezoarticus leucogaster) of the Brazilian Pantanal

“…As well as being infected by A. marginale, the cervids are also infected with Anaplasma phagocitophylum (DUMLER et al, 2001) and Anaplasma ovis (HAIGH et al, 2008), which makes it difficult to implement a surveillance by serology. The current serological tests display crossed reactions, even when antigenic sub-units such as MSP5 recombinant protein are employed as antigens, because Anaplasma species have similar B epitopes (STRIK et al, 2007).…”