DIFFERENCES AND SIMILARITIES BETWEEN Anaplasma marginale and Anaplasma phagocytophylum
DIFERENCIAS Y SIMILITUDES ENTRE Anaplasma marginale y Anaplasma phagocytophilum
Belkis Corona and Siomara Martínez
National Centre for Animal and Plant Health (CENSA), Apartado 10, San José de las Lajas, La Habana, Cuba. E-mail: bcorona@censa.edu.cu ]]>
ABSTRACT
Anaplasma marginale and A. phagocytophilum are intracellular rickettsial pathogens causing bovine anaplasmosis and human granulocytic anaplasmosis, respectively. Effective vaccines for the control of anaplasmosis are not available despite attempts using different approaches, such as attenuated strains, infected erythrocyte and tick cell-derived purified antigens. Recent reports demonstrated that A. marginale and A. phagocytophilum co-exist in geographic areas, that concurrent infections may occur in ruminants and ticks and that there are similarities and differences at molecular level between both species. The aim of this article is to make a comparison between the main characteristics of Anaplasma marginale and Anaplasma phagocytophilum.
Key words: Anaplasma marginale; Anaplasma phagocytophilum; major surface proteins
RESUMEN
Anaplasma marginale y A. phagocytophilum son rickettsias intracelulares que causan anaplasmosis bovina y anaplasmosis granulocítica humana, respectivamente. Aún no existe una vacuna efectiva para el control de la anaplasmosis, a pesar de haber sido utilizados con este fin cepas atenuadas, eritrocitos infectados y antígenos purificados, derivados de células de garrapatas. Reportes recientes han demostrado que A. marginale y A. phogocytophilum coexisten en áreas geográficas, que infecciones concurrentes pueden ocurrir en rumiantes y garrapatas y que existen similitudes y diferencias a nivel molecular entre estos dos microorganismos. El objetivo de este trabajo es realizar una comparación entre las principales características de A. marginale y A. phagocytophilim.
Palabras clave: Anaplasma marginale; Anaplasma phagocytophilum; proteínas principales de la superficie
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A. marginale is a rickettsial organism causing bovine anaplasmosis in cattle with significant economic losses in tropical and subtropical regions worldwide. It invades the erythrocyte and leads to extravascular hemolysis. Ticks are biological vectors of A. marginale but the pathogen is often transmitted mechanically to susceptible cattle by blood-contaminated mouthparts of biting flies or fomites. These obligate intracellular organism replicates in membrane-bound parasitophorous vacuoles in bovine erythrocytes or tick cells. Both cattle and ticks become persistently infected with A. marginale and thus serve as reservoirs of infection (1).Many geographic strains of A. marginale have been identified, which differ in biology, genetic characteristics and transmissibility by ticks. The genetic diversity of A. marginale strains have been characterized using major surface protein (MSP) genes involved in interactions with vertebrate host cells (2). These genes may have evolved more rapidly than other genes because of selective pressures exerted by the host immune system (3). Some studies have demonstrated genetic variation among different A. marginale strains (4), by means of Random Amplified Polymorphic DNA (5); restriction fragment length polymorphism analysis (6), Repetitive Extragenic Consensus (REP/ERIC) PCR patterns (4), PCR assay based on specific sequences of MSP (7;8) and protein sequences (9).
de la Fuente et al. (10) results support the hypothesis that genetic heterogenicity observed among strains of A. marginale within endemic regions could be explained by cattle movement and maintenance of different genotypes by independent transmission events, due to infection exclusion of A. marginale in cattle and ticks, which commonly results in the establishment of only one genotype per animal. However, when distantly related genotypes exist in the same region, infections of a single host with multiple A. marginale strains are possible (11).
The presence of different A. marginale genotypes in different countries (12) suggests that MSP1a sequences, although conserved during multiplication of the parasite in the bovine host and after transmission by ticks (13), are rapidly changing, resulting in genotype variation within A. marginale populations. For this reason the DNA sequence of the msp1a gene does not provide a distinct phylogeographical resolution, because of its high variability (7;8). In contrast, msp4 sequences may provide useful phylogeographical information (9).
Anaplasma marginale can persist in ruminants host for the animal whole life (14). In cattle persistently infected with A. marginale, there are cyclic peaks of rickettsemia every 2 to 6 weeks containing different variants of the immunoprotective major surface protein MSP2 (15). MSP2 is encoded by a multigene family and sequence variation is achieved by segmental gene conversion of a single polycistronic expression site by different pseudogenes. These pseudogenes contain a hypervariable region and portions of flanking 5´and 3´conserved sequences but they are otherwise truncated and cannot encode full-length MSP2 (16).
Membrane surface proteins can be useful as microbial identifiers and therefore act as antigens eliciting an immune response. Safe vaccine possibilities currently include testing if recombinant major surface protein antigens produce an effective immune response to protect the animal from future infections (17).
Major surface protein 5 (MSP5) of Anaplasma marginale was determined to be antigenic and highly
conserved among various isolates of A. marginale (5;18). Antibodies to MSP5 of A. marginale were recognized in both acute stages of infection and chronically infected carrier cattle, a highly sensitive and specific competitive ELISA was developed using this antigen and a monoclonal antibody to MSP5 (19); for these reasons the msp5 gene and the MSP5 protein are the best candidates for Anaplasma marginale diagnosis (20).
Anaplasma phagocytophilum
The order Rickettsiales represents an 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 (21). 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 now reclassified as Anaplasma phagocytophilum, the causative agent of granulocytic anaplasmosis, an emerging tick-borne disease in the United State and Europe (22). Closely related to Ehrlichial and Rickettsial organisms, A. phagocytophilum is a small, fragile, Gram-negative bacterium presenting unique challenge for culture, isolation, enumeration, and labelling (23;24). A. phagocytophilum has been worldwide detected, particularly in North America and Europe as well as in South Africa, South America, and Asia (25). ]]>
Infection with A. phagocytophilum has been recognized in a variety of mammalian hosts, including humans, cats, dogs, horses, ruminants, and wildlife species (26). Clinical disease ranges from mild to fatal, and associated findings include fever, anorexia, weight loss, polyarthritis, and possibly meningitis. Laboratory findings most often include thrombocytopenia and/or lymphopenia and neutropenia. Current methods of serologic diagnosis primarily involve the use of an indirect immunofluorescent antibody test (IFA).The organisms are transmitted by ticks and their life cycle varies with the type of tick species, tick population density, and the wildlife that are indigenous to an specific climate and geographic location. Wildlife animals such as deer, rodents, and other small mammals maintain A. phagocytophilum and serve as the reservoir hosts, with transmission to domestic animals and man as a result of tick bites (27).
The outer membrane proteins of A. phagocytophilum have not been characterized systematically. The Omp-1/P44/Msp2 superfamily is the most studied outer membrane protein family of A. phagocytophilum. The genome has three omp-1, one msp2, two msp2 homolog, one msp4, and 113 p44 loci encoding proteins belonging to this superfamily (28).
Recent A. phagocytophilum genome sequencing data have provided a wealth of new genetic information. However, there is no experimental evidence demonstrating A. phagocytophilum surface-exposed proteins in addition to P44. Furthermore, almost one-half of the predicted open reading frames of A. phagocytophilum encodes conserved or novel hypothetical proteins that have never been characterized in any bacterium, some of which may be surface proteins. Therefore, it is imperative to use new approaches, including proteomics, to generate a more complete picture of the expression and function of A. phagocytophilum surface proteins (28).
The PCR detection of A. phagocytophilum DNA is both sensitive and specific for the acute phase diagnosis of anaplasmosis (29). With the advent of real-time PCR and with the increased availability of molecular diagnostic testing, veterinarians should be able to obtain rapid confirmation of the diagnosis of anaplasmosis.
Differences and similarities between Anaplasma marginale and Anaplasma phagocytophilum.
Anaplasma marginale and A. phagocytophilum are closely related, obligate intracellular, tick-borne rickettsial animal pathogens that parasitize two very different host cell types. Only ruminants are known to be susceptible to A. marginale. Whereas the spectrum of species infected with the zoonotic A. phagocytophilum includes small rodents, ruminants, dogs, horses and humans (21).
It is thought that A. marginale exclusively infect bovine erythrocytes. Acute bovine erythrocytic anaplasmosis is characterized by severe anemia, icterus and hemoglobinuria due to removal of infected cells. Animals recovered remain chronically infected for life, and experience regular, but low level of parasitemia (30). Each rickettsemia peak is composed of new antigenic variants that are selected following a specific antibody responses by the host. By contrast, A. phagocytophilum infections produce an acute, febrile illness accompanied by appearance of characteristic colonies of the microbes in peripheral blood neutrophil granulocytes as well as their precursors in the bone marrow (31) with concomitant impairment of resistance to secondary infection.
A. phagocytophilum can be cultured in vitro in a human promyelocytic cell line, HL-60, as well as in cell lines ISE6 and IDE8 from the North American tick vector, Ixodes scapulari (32;33). A. marginale can likewise be propagated in Ixodes tick cell lines. However, a continuous mammalian culture system has been lacking for Anaplasma marginale, and the corresponding cell surface receptors are unknown. Moreover, no nucleated host cells of A. marginale have been identified, and none of the cell lines that support A. phagocytophilum could be infected with A. marginale (34).
There are similarities at the molecular level between A. margianle and A. phagocytophilum. Similar to A. marginale, A. phagocytophilum uses combinatorial mechanisms to generate a large array of outer membrane protein variants. Such gene polymorphism has profound implications for the design of vaccines, diagnostic test and therapy (30). ]]>
In Anaplasma marginale, persistence is associated with antigenic variation of the immunoprotective outer membrane protein MSP2. Extensive diversity of MSP2 is achieved by combinatorial gene conversion of a genomic expression site by truncated pseudogenes. The major membrane protein of A. phagocytophilum, MSP2 (P44), homologous to MSP2 of Anaplasma marginale, has a similar organization of conserved and variable regions, and it is also encoded by a multigene family containing some truncated gene copies (16). This suggests that the two organisms could use similar mechanisms to generate diversity in outer membrane proteins from their small genomes.As in A. marginale infections, a dominant antibody response in patients infected with A. phagocytophilum is expressed against a variable 40 kDa outer membrane protein MSP2 (P44) (35). The gene encoding MSP2 (P44), like msp2, is a member of a cross-hybridizing multigene family and it is homologous to A. marginale msp2 (60 to 66% similarity and 40 to 53% identity), depending of the gene and the strain. Sequence alignment of different msp2 (p44) variants and A. marginale msp2 reveals significant variation in the same central hypervariable region (36). As in A. marginale, the A. phagocytophilum genome contains incomplete msp2 (p44) genes with a unique central hypervariable region and conserved 5´and 3´ flanking sequences (37) that could be a source of diversity for combinatorial recombination mechanisms. The results obtained by Barbet et al. (30), related with MSP2 (P44) from A. phagocytophilum and MSP2 from A. marginale suggest that similar mechanisms for generating outer membrane protein diversity and establishing infections are available to the two organisms.
A. marginale St. Maries is reported to have 56 genes that have been placed into this superfamily, including eight msp2, eight msp3, one msp4, three opag, 15 omp-1, 12 orfX, seven orfY, and two msp3 remnants. These genes are scattered throughout the genome with a bias in location towards the origin of replication. MSP2 and MSP3 are the immunodominant proteins. The msp2 and msp gene subsets each include one full-length expression locus and seven reserve/silent sequences that are thought to recombine into the expression locus to generate antigenic variation (38).
The A. phagocytophilum genome has three omp-1, one msp2, two msp2 homolog, one msp4, and 113 p44 loci belonging to the OMP-1/MSP2/P44 superfamily. Although both Anaplasma spp. msp2 genes are members of PF01617 and the OMP1/MSP2/P44 superfamily, the A. marginale msp2 gene is distinct from the A. phagocytophilum msp2 gene. In addition, the previously identified omp-1N is not a member of this Pfam, but it is homologous to E. chaffeensis omp-1N and the msp2 operon-associated gene 3 of A. marginale (39).
Other outer membrane proteins have been reported in A. marginale, including msp5, msp1a, and msp1b. The msp5 gene (a SCO1/SenC family protein) is found in all the Rickettsiales, whereas msp1a and msp1b are unique to A. marginale. Sequence homologies have also been shown for the MSP4 gene of the two pathogens (2).
Even before the recent reclassification within the family Anaplasmataceae, the msp5 gene was known to be highly conserved among all Anaplasma species, (11) which, at that time, included A. marginale, A. centrale, and A. ovis (18). Based on 16S rRNA gene sequence similarity, A. phagocytophilum and A. platys were placed within the same family (21). Strik et al. (25) demonstrated the high conservation of MSP5 of A. phagocytophilum among various isolates in the United State and Europe. Because of the cross-reactivity between the MSP5 orthologs of A. phagocytophilum and A. marginale, the commercial available cELISA should be used in epidemiological studies where distinctions between these two infectious agents in cattle are necessary. However, MSP5 or MAP2 might serve as a screening tool for the rapid clinical diagnosis of ehrlichiosis and/or anaplasmosis in various species.
Recombinant MSP5 of A. marginale has been used as a diagnostic-test antigen in an indirect-ELISA format
for surveying A. marginale infection in cattle in several countries. The results obtained by Alleman et al. (40) indicate that an indirect ELISA using rMSP5 of A. marginale cannot distinguish between infections with A. phagocytophilum and infections with A. marginale. Cattle infected with either organism will likely be seropositive, giving rise to false representation of disease incidence in a particular area.
A major protein antigen(s) is expressed on the outer membrane of A. phagocytophilum, and some of the immunodominant major surface proteins (MSPs) share sequence similarity with Anaplasma marginale MSP2 and MSP4 (41;42) and Ehrlichia ruminantium MAP1 (43). Although the biological function of A. marginale MSP4 is unknown, this MSP is probably involved in host-pathogen interactions and may evolve more rapidly than other nuclear gene proteins because of selective pressures exerted by host immune systems. Furthermore, the analysis of msp4 sequences provided phylogeographic patterns for A. marginale strains (9).
Although A. phagocytophilum has a broad geographic distribution, all strains identified thus far appear to have considerable serological cross-reactivity and a minor degree of variation in the nucleotide sequences of the 16S rRNA, groESL, gltA, ank, and msp2 genes (44), with the exception of some ank sequences from infected German ticks that are different from other ank sequences of human and animal strains (21). However, the clinical and host tropism diversity of A. phagocytophilum suggests the presence of genetic differences among these bacteria that have not been characterized. ]]>
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(Recibido 2-8-2008; Aceptado 21-12-2008) ]]>