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PubMed2000

Antigenic variation of Anaplasma marginale by expression of MSP2 mosaics.

Anaplasma marginale is a tick-borne pathogen, one of several closely related ehrlichial organisms that cause disease in animals and humans. These Ehrlichia species have complex life cycles that require, in addition to replication and development within the tick vector, evasion of the immune system in order to persist in the mammalian reservoir host. This complexity requires efficient use of the small ehrlichial genome. A. marginale and related ehrlichiae express immunoprotective, variable outer membrane proteins that have similar structures and are encoded by polymorphic multigene families. We show here that the major outer membrane protein of A. marginale, MSP2, is encoded on a polycistronic mRNA. The genomic expression site for this mRNA is polymorphic and encodes numerous amino acid sequence variants in bloodstream populations of A. marginale. A potential mechanism for persistence is segmental gene conversion of the expression site to link hypervariable msp2 sequences to the promoter and polycistron.

Amino Acid Sequence↗
PubMed2003

Characterization of Anaplasma marginale isolated from North American bison.

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.

Amino Acid Sequence↗
PubMed2003

[Prevalence of Anaplasma marginale and specific antibodies in new born calves].

Anaplasma marginale is an intraerythrocytic rickettsia that affects the cattle and constitute a constraint for its production. The transplacental transmission of A. marginale could be of importance under field conditions, but until now no data is available to interpret its relevance in Venezuela. The goal of this work was to evaluate both the presence of A. marginale and the antibody response against the rickettsia in new born calves. Fourteen calves, one week old, were simultaneously bled for parasitological tests, packed cell volume measurement and to obtain sera for immunoenzymatic assays. All the animals showed parasitemia between seventeen and thirty days old. The antibody IgM and IgG response at this time was low. Probably, these calves acquired A. marginale infection through transplacental route and a very low level of immunity by the colostrum.

Anaplasma marginale↗
PubMed1979

In vitro cultivation of Anaplasma marginale: growth pattern and morphologic appearance.

Anaplasma marginale propagated in vitro showed an increasing rate of replication in a sequence of three experiments. The changes in the percentage of parasitized erythrocytes and identification of the organism in culture were monitored by the Giemsa-staining and the direct fluorescent antibody techniques. The ultrastructure of the organism in culture also was determined. The percentage of parasitized erythrocytes increased more than three times in the first experiment during a period of 8 days, and about ten times in the second experiment during a period of 14 days. After 8 days of observation of the primary culture in the third experiment, the trend of growth was more rapid than in the first and second experiment. The viability of the organism was verified by inoculation of susceptible calves, using 13- and 33-day cultures of the first and the second experiments, respectively. The primary cultures were subcultured twice by dilution with normal bovine erythrocytes.

Anaplasma↗
PubMed2006

Analysis of world strains of Anaplasma marginale using major surface protein 1a repeat sequences.

Anaplasma marginale is a tick-borne pathogen of cattle that causes the disease bovine anaplasmosis worldwide. Major surface proteins (MSPs) are involved in host-pathogen and tick-pathogen interactions and have been used as markers for the genetic characterization of A. marginale strains and phylogenetic studies. MSP1a is involved in the adhesion and transmission of A. marginale by ticks and varies among geographic strains in the number and sequence of amino-terminal tandem repeats. The aim of this study was to characterize the genetic diversity of A. marginale strains collected from countries in North and South America, Europe, Asia, Africa and Australia, inclusive of all continents. In this study, we characterized 131 strains of A. marginale using 79 MSP1a repeat sequences. These results corroborated the genetic heterogeneity of A. marginale strains in endemic regions worldwide. The phylogenetic analyses of MSP1a repeat sequences did not result in clusters according to the geographic origin of A. marginale strains but provided phylogeographic information. Seventy-eight percent of the MSP1a repeat sequences were present in strains from a single geographic region. Strong (> or =80%) support was found for clusters containing sequences from Italian, Spanish, Chinese, Argentinean and South American strains. The phylogenetic analyses of MSP1a repeat sequences suggested tick-pathogen co-evolution and provided evidence of multiple introductions of A. marginale strains from various geographic locations worldwide. These results contribute to the understanding of the genetic diversity and evolution of A. marginale and tick-pathogen interactions.

Amino Acid Sequence↗
PubMed1996

Establishment of the tick (Acari:Ixodidae)-borne cattle pathogen Anaplasma marginale (Rickettsiales:Anaplasmataceae) in tick cell culture.

Anaplasma marginale is a tick-borne rickettsia that causes bovine anaplasmosis worldwide. Despite its importance, A. marginale has thus far not been established in a continuous culture system. We have propagated A. marginale continuously for the 1st time in a tick cell line derived from the black-legged tick, Ixodes scapularis Say, using infected bovine blood as the inoculum. Erythrocytic stages invaded the tick cells and multiplied in membrane-lined vacuoles to form colonies typical of those observed in naturally infected ticks as demonstrated by light and electron microscopy. The rickettsiae have been passaged serially for 3 yr and have been cryopreserved in liquid nitrogen. Antigens present in A. marginale from tick cell culture were recognized by bovine immune serum against the blood stages of A. marginale. A. marginale grown in this tick cell line was infective for calves, and male ticks fed on the calves transmitted A. marginale to a susceptible calf. The ability to culture A. marginale removes a major impediment to the study of Anaplasma biology in vitro, and will enhance development of vaccines and diagnostic tests.

Anaplasma↗
PubMed1984

Immune serum against Anaplasma marginale initial bodies neutralizes infectivity for cattle.

The first demonstration of immune serum-mediated neutralization of Anaplasma marginale and identification of the involved surface antigens is presented. Anaplasma marginale initial bodies were purified from parasitized erythrocytes by using ultrasonic disruption and differential centrifugation. The initial bodies were morphologically intact, as determined by electron microscopy, were not agglutinated by anti-bovine erythrocyte sera, and were infective. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated minimal erythrocytic contamination in the purified initial bodies. Rabbit antisera against initial bodies completely neutralized the infectivity of purified initial bodies for splenectomized calves, whereas normal rabbit serum did not alter infectivity. Nine initial body proteins were surface radioiodinated by using lactoperoxidase, and five of these were precipitated by the neutralizing antibody (105,000, 86,000, 61,000, 36,000, and 31,000 apparent m.w.). The results demonstrate that initial body determinants capable of inducing neutralizing antibody are present. The identification of surface radiolabeled proteins immunoprecipitated by the neutralizing antibody provides candidates to be tested as protective immunogens in cattle.

Anaplasma↗
PubMed2000

Sequence and expression analysis of a surface antigen gene family of the rickettsia Anaplasma marginale.

The tick-borne rickettsial organism, Anaplasma marginale, causes a disease in cattle of world-wide economic significance. This disease, anaplasmosis, is characterized by severe hemolytic anemia, high levels of rickettsemia and, often, death in animals over 3years of age. Animals that survive acute infection remain carriers, with continuous sub-microscopic cycles of rickettsemia that can persist for the lifetime of the animal. In the search for potential recombinant immunogens, it was discovered that several surface proteins of A. marginale encode polymorphic multigene families. Despite the small size of the genome (approx. 1250kb), these surface antigen gene families comprise greater than 2% of the genome. We present here a mapping, sequencing and expression analysis of five complete or partial genes encoding MSP1b in a Florida strain of A. marginale. Two genes are complete; they encode mRNA that is translated into polypeptide products. Three genes are incomplete and appear to be derived from the complete genes by a series of segmental intragenic recombinations. In two of the incomplete genes, 5' sequence in the incomplete genes is 3' sequence in the complete genes. Recombination within these gene families may generate diversity in surface antigens through combinatorial rearrangements. This could contribute to persistence in the chronic infections caused by A. marginale and related rickettsiae.

Anaplasma↗
PubMed1979

Testicular degeneration and libido loss in beef bulls experimentally inoculated with Anaplasma marginale.

Four sexually mature virgin beef bulls were inoculated with whole blood from a known Anaplasma marginale carrier. All four bulls became infected and experienced some degree of anemia and testicular degeneration, while uninoculated controls remained normal. Testicular degeneration was confirmed by electron microscopy, histopathology, and semen evaluation. Further observations established that the inoculated bulls exhibited a loss of libido, while controls reacted in a normal manner to a cow in estrus. Anaplasma marginale was not present in semen of inoculated bulls.

Journal Article↗
PubMed1993

Analysis of the Anaplasma marginale genome by pulsed-field electrophoresis.

Anaplasma marginale is a rickettsial parasite of bovine erythrocytes causing world-wide economic losses in livestock production. Despite its importance, little is known about this rickettsia at a molecular level because it has not been cultured in vitro, and there is no small-animal model. Although several genes have been cloned and sequenced, the gross genome structure of the organism has not yet been well characterized. We separated intact bovine erythrocytes from leucocytes, and determined the genome size of A. marginale by use of restriction endonuclease cleavage and pulsed-field gel electrophoresis (PFGE). A value of 56 mol% G+C was obtained for this genome by spectral analysis. Undigested A. marginale DNA failed to migrate under several different electrophoretic conditions, indicating a circular genome. Digestions of intact A. marginale DNA were performed using restriction endonucleases NotI, SfiI and PacI. Complete digestion with SfiI resulted in 12 distinct bands ranging in size from 14 to 170 kbp. Total size determined by addition of SfiI-digested fragments was approximately 1200 kbp. PacI cleaved the A. marginale genome from three different isolates into just three fragments, of 598, 557 and 97 kbp. Incomplete digestion produced a band measuring 1250 kbp. These results indicate that A. marginale has a circular genome between 1200 and 1260 kbp, with a G+C content of 56 mol%.

Anaplasma↗
PubMed1993

Impact of persistent Anaplasma marginale rickettsemia on tick infection and transmission.

Anaplasma marginale, an intraerythrocytic rickettsia of cattle, is transmitted biologically by ticks. Because of the brevity of acute A. marginale infection, transmission may rely on the tick's ability to acquire the organism from persistently infected cattle with low rickettsemia levels. By using a nucleic acid probe to quantitate low-level infection, we found that rickettsemia levels in persistently infected cattle fluctuated at approximately 5-week intervals during a 24-week period, from < 10(4) infected erythrocytes per ml of blood to high levels of approximately 10(7) infected erythrocytes per ml of blood. Cattle maintained very low rickettsemia levels (< 10(4.3) infected erythrocytes per ml of blood) for approximately 4 to 8 days of every 5-week cycle. The effect of fluctuations in rickettsemia in persistently infected cattle on acquisition by Dermacentor andersoni nymphal and adult male ticks was examined. A positive correlation was observed between rickettsemia levels in cattle and the resulting infection rates of ticks. At high rickettsemia levels, up to 80% of ticks acquired infection, but even at extremely low rickettsemia levels, 27% of adult male ticks became infected. Moreover, once ticks acquired infection, biological replication of the organism within the ticks appeared to make up for initial differences in the infecting dose. The high infection rates in adult males, combined with their intermittent feeding behavior and the observation that only a few infected ticks were required for transmission to a susceptible host, suggest that adult male D. andersoni ticks are epidemiologically important in A. marginale transmission. Because cattle with all levels of rickettsemia were capable of efficient transmission to ticks, population control efforts must include decreasing transmission from persistently infected individuals.

Anaplasma↗
PubMed2001

Differential adhesion of major surface proteins 1a and 1b of the ehrlichial cattle pathogen Anaplasma marginale to bovine erythrocytes and tick cells.

Anaplasma marginale is a tick-borne ehrlichial pathogen of cattle for which six major surface proteins (MSPs) have been described. The MSP1 complex, a heterodimer composed of MSP1a and MSP1b, was shown to induce a protective immune response in cattle and both proteins have been identified as putative adhesins for bovine erythrocytes. In this study the role of MSP1a and MSP1b as adhesins for bovine erythrocytes and tick cells was defined. msp1alpha and msp1beta1 genes from the Oklahoma isolate of A. marginale were cloned and expressed in Escherichia coli K-12 under the control of endogenous and tac promoters for both low and high level protein expression. Expression of the recombinant polypeptides was confirmed and localised on the surface of transformed E. coli. The adhesion properties of MSP1a and MSP1b were determined by allowing recombinant E. coli expressing these surface polypetides to react with bovine erythrocytes, Dermacentor variabilis gut cells and cultured tick cells derived from embryonic Ixodes scapularis. Adhesion of the recombinant E. coli to the three cell types was determined using recovery adhesion and microtiter haemagglutination assays, and by light and electron microscopy. MSP1a was shown by all methods tested to be an adhesin for bovine erythrocytes and both native and cultured tick cells. In contrast, recombinant E. coli expressing MSP1b adhered only to bovine erythrocytes and not to tick cells. When low expression vectors were used, single E. coli expressing MSP1a was seen adhered to individual tick cells while reaction of tick cells with the E. coli/MSP1a/high expression vector resulted in adhesion of multiple bacteria per cell. With electron microscopy, fusion of E. coli cell membranes expressing MSP1a or MSP1b with erythrocyte membranes was observed, as well as fusion of tick cell membranes with E. coli membranes expressing MSP1a. These studies demonstrated differential adhesion for MSP1a and MSP1b for which MSP1a is an A. marginale adhesin for both bovine erythrocytes and tick cells while MSP1b is an adhesin only for bovine erythrocytes. The role of the MSP1 complex, therefore, appears to vary among vertebrate and invertebrate hosts.

Adhesins, Bacterial↗
PubMed1964

IMMUNOLOGY AND SEROLOGY OF ANAPLASMA MARGINALE. I. FRACTIONATION OF THE COMPLEMENT-FIXING ANTIGEN.

Rogers, Thomas E. (Louisiana State University, Baton Rouge), Richard J. Hidalgo, and George T. Dimopoullos. Immunology and serology of Anaplasma marginale. I. Fractionation of the complement-fixing antigen. J. Bacteriol. 88:81-86. 1964.-Studies were conducted to fractionate and purify the complement-fixation (CF) antigen of Anaplasma marginale in infected erythrocytes of cattle. Initial attempts were made to resolve the antigen from crude stromatal preparations by various chemical and physical methods. Fractionation procedures involving partial and total lipid extraction suggested that the CF antigen was lipoprotein in nature. Fluorocarbon deproteinization of stromatal antigens was also attempted. A method was developed for the preparation of a desirable Anaplasma CF antigen which involved disintegration of infected erythrocytes by sonic vibration and separation of the antigen by differential centrifugation. Antigens prepared by this method were highly specific, colorless, did not exhibit anticomplementary activity, and possessed higher titers than standard Anaplasma antigens. When density-gradient sedimentation was applied to sonic extracts of infected cells, it was demonstrated that the CF antigen could also be fractionated by this method.

Anaplasma↗
PubMed1980

Susceptibility of Bos indicus crossbred and Bos taurus cattle to Anaplasma marginale infection.

The effects of infection with Anaplasma marginale in groups of Bos indicus-Brahman crossbred and Bos taurus cattle aged 27 and 48 months were studied using the parameters of body weight change, packed cell volume, parasitaemia and humoral antibody. Twenty-six animals (8 Bos taurus and 18 Bos indicus crosses) of each age group were infected. No significant differences between any of the groups were observed.

Anaplasma↗
PubMed1990

Cyclic rickettsemia during persistent Anaplasma marginale infection of cattle.

Submicroscopic levels of Anaplasma marginale rickettsemia in persistently infected cattle were determined by using nucleic acid hybridization. Within individuals, the rickettsemia levels steadily increased from less than 10(4) infected erythrocytes per ml to a peak of more than 10(6) infected erythrocytes per ml and then rapidly declined. This logarithmic variation parallels the variation of the rickettsemia level seen in acute infection and suggests that cyclic emergence of antigenic variants is a mechanism of rickettsial persistence.

Anaplasma↗
PubMed1967

Evidence of lactate dehydrogenase in Anaplasma marginale.

Extracts from preparations of partially purified Anaplasma marginale revealed low levels of lactate dehydrogenase (LDH). Enzyme inhibition by immune sera further indicated that A. marginale possesses a protein moiety the same as that of the normal red blood cell (RBC), although data suggested an alteration of LDH(1) from that observed in normal RBC. Bimodal isozyme distribution was detected after electrophoresis of the extracts. One isozyme approached the cathode and the other the anode, and both appeared to be nicotinamide adenine dinucleotide-dependent. Heterogeneity of parasite and host cell isozymes was established on the basis of zone electrophoresis on cellulose acetate strips.

Anaplasma↗
PubMed1975

Propagation of Anaplasma marginale in bovine lymph node cell culture.

Anaplasma marginale was propagated in cell cultures derived from bovine lymph node (LN). Treatment of host cells with diethylaminoethyl dextran (DEAE-D) before inoculation and centrifugation of inoculum onto the monolayers resulted in significant numerical increases of A marginale. The direct fluorescent antibody technique (FAT) was used for detection of the organism in culture. The rat was combined with the standard microscopic count procedure to obtain numerical estimates of the organism in cell culture. Infection of LN cells was irregular, with some cells containing many organisms and others containing none. The organisms were dispersed or in inclusions in the cytoplasm of LN cells. Numerical increases of organisms occurred within 6 hours and these were greatest at 12 to 24 hours. After 24 hours, the organisms decreased rapidly, but small numbers of them were observed for at least 7 days. The average generation time in culture was approximately 17.1 hours.

Anaplasma↗
PubMed2003

Infection exclusion of the rickettsial pathogen anaplasma marginale in the tick vector Dermacentor variabilis.

Anaplasma marginale is a tick-borne, rickettsial cattle pathogen that is endemic in several areas of the United States. Recent studies (J. de la Fuente, J. C. Garcia-Garcia, E. F. Blouin, J. T. Saliki, and K. M. Kocan, Clin. Diagn. Lab. Immunol. 9:658-668, 2002) demonstrated that infection of cultured tick cells and bovine erythrocytes with one genotype of A. marginale excluded infection with other genotypes, a phenomenon referred to as infection exclusion. The present study was undertaken to confirm the phenomenon of infection exclusion of A. marginale genotypes in a tick vector, Dermacentor variabilis. Only one genotype of A. marginale (Virginia isolate) was detected by PCR in ticks that fed first on a calf infected with a Virginia isolate and second on a calf infected with an Oklahoma isolate. These studies demonstrate that infection exclusion of A. marginale genotypes also occurs in naturally infected ticks, as well as in cattle and cultured tick cells, and results in establishment of only one genotype per tick.

Anaplasmataceae↗