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PubMed1989

Infectivity and antigenicity of Anaplasma marginale from tick cell culture.

The infectivity and immunogenicity of Anaplasma marginale grown in a tick cell culture from embryonic Dermacentor variabilis ticks were assessed in splenectomized and intact calves, respectively. Culture 1 consisted of the cell line inoculated with midguts of adult ticks infected with the Mississippi isolate of A marginale and dissected 5 to 10 days after repletion and detachment from an experimentally infected calf. Cultures 2 and 3 consisted of the cell line inoculated with midguts of ticks infected with the Virginia isolate of the organism. Inoculum for culture 2 was derived from nymphal ticks dissected 5 to 10 days after repletion and detachment from the infected calf; inoculum for culture 3 was midguts from adult ticks that were fed as nymphs, allowed to molt in the laboratory and dissected 21 to 24 days after molting. In trial 1, cultures 1, 2, and 3 were maintained at pH 6.9 and incubated at 28 C; in trial 2, cultures 1 and 3 were maintained at pH 7.4 and incubated at either 28 C or 37 C. Cultures 1, 2, and 3 failed to induce infection when injected IV and SC into 6 calves in 2 separate trials. Pre-challenge sera from these calves reacted with 2 purified Anaplasma antigens in the ELISA, but failed to react in the complement-fixation test. Results of a trial to use cultures 1 and 3 in combination with an oil-in-water adjuvant to immunize intact calves against A marginale were inconclusive. However, pre-challenge sera from immunized calves reacted with the 2 purified Anaplasma initial body antigens in the ELISA but failed to react in the complement-fixation text.(ABSTRACT TRUNCATED AT 250 WORDS)

Anaplasma↗

PubMed2002

Effect of tetracycline on development of Anaplasma marginale in cultured Ixodes scapularis cells.

Infections of the tick-borne ehrlichial pathogen, Anaplasma marginale, in cattle have been controlled, in part, by administration of low doses of tetracycline. Recently, a cell culture system was developed for A. marginale using a tick cell line derived from embryonic Ixodes scapularis. This study was designed to determine the effect of tetracycline on A. marginale propagated in a tick cell culture assay. Various concentrations of tetracycline (0, 0.01, 0.10, 1.0, 5, 10, 20 or 100 microg/ml) were added in medium to cultures 48h after cell monolayers were inoculated with A. marginale. A. marginale growth in the drug treated and control cultures was subsequently evaluated by indirect ELISA at 7 days post-infection (PI) and daily by light and electron microscopy (LM and EM). Infectivity of the culture-derived A. marginale was determined by inoculation of susceptible cattle with treated and untreated control cultures. Tetracycline doses of 5, 10, 20 and 100 microg/ml resulted in significant inhibition of A. marginale growth as determined by ELISA. Morphologic deterioration of Anaplasma, as determined by LM and EM, occurred in cultures treated with the same drug concentrations. A. marginale replication, inhibited in cultures treated on days 2-6 PI with 20 microg/ml tetracycline, was not apparent 96 days after antibiotic removal. Infected cell cultures treated with medium containing 20 microg/ml tetracycline proved to be non-infective when inoculated into susceptible splenectomized calves. All parameters studied herein demonstrated that tetracycline killed A. marginale in cultured tick cells. The Anaplasma-tick cell culture drug assay therefore, would be useful for screening and evaluating novel antibiotics for control of anaplasmosis.

Anaplasma↗

PubMed1986

Longevity of colonies of Anaplasma marginale in midgut epithelial cells of Dermacentor andersoni.

Colonies of Anaplasma marginale in midgut epithelial cells of experimentally infected Dermacentor andersoni were studied in adult ticks 1, 3, and 6 months old. Longevity of the parasite in ticks was assessed by evaluating its infectivity for splenectomized calves; calves were exposed by feeding ticks and by inoculation of tick gut homogenates. Longevity was also evaluated by determining size, type, and density of colonies in male and female ticks. The effect of incubation (2.5 days at 37 C) on colony density was also examined for ticks at each age period. All methods used to assess longevity of A marginale in ticks (tick transmission, calf inoculation, and histologic studies) indicated a decrease of the numbers of organisms in 6-month-old ticks. Furthermore, when tick gut homogenates from 6-month-old nonincubated ticks were not infectious for susceptible calves, incubation of ticks before dissection restored infectivity of homogenates. Colonies of A marginale were detected in gut tissues of 6-month-old ticks that were not infective; therefore, infectivity of ticks could not be confirmed merely by presence of A marginale colonies.

Anaplasma↗

PubMed1999

Dermacentor hunteri (Acari: Ixodidae): an experimental vector of Anaplasma marginale and A. ovis (Rickettsiales: Anaplasmataceae) to calves and sheep.

The experimental vector competence of laboratory-reared Dermacentor hunteri Bishopp for Anaplasma marginale Theiler and Anaplasma ovis Lestoquard was evaluated by delayed transfer of male ticks from infected to susceptible Holstein calves and from infected to susceptible domestic sheep, respectively. After feeding for 4 or 5 d on rickettsemic acquisition hosts, the ticks were held off the host at 26 degrees C, approximately 93% RH, and a photoperiod of 14:10 (L:D) h for 7 or 8 d, then test fed for 5 or 7 d. Additionally, ticks test-fed for 5 d on 2 susceptible calves were removed, held off the host for 7 d, and test-fed for 5 d on a 3rd susceptible calf to test the tick's ability to transmit A. marginale by delayed serial transfer. Tick transmission of A. marginale to 3 test calves and A. ovis to 3 test sheep was demonstrated by blood smear and indirect immunofluorescence serology. These data indicate that males of D. hunteri, a tick commonly found on desert bighorn, Ovis canadensis Shaw, in the southwestern United States and northern Mexico, may be competent natural vectors of these organisms present in desert bighorn populations.

Anaplasma↗

PubMed1975

Morphologic alterations of Anaplasma marginale in calves after treatment with oxytetracycline.

The morphologic features of Anaplasma marginale were determined after treatment of infected calves with oxytetracycline. By light microscopy, anaplasma bodies in erythrocytes of treated calves were enlarged and vacuolated, small and dense, or comma shaped. Several degenerated forms of anaplasma bodies were observed by electron microscopy. There was a blending of the 2 membranes surrounding initial bodies, aggregation of nucleoprotein at the periphery of the subunit, and vacuolation. A 2nd form of degeneration was coalescence of subunits in marginal bodies. A 3rd form of degeneration was unification of subunits of anaplasma bodies and clumping of nucleoprotein. A 4th form of degeneration was the persistence of an anaplasma body as a solitary, irregularly shaped mass containing electron-opaque clumps.

Anaplasma↗

PubMed2002

Antigenic variation of Anaplasma marginale msp2 occurs by combinatorial gene conversion.

The rickettsial pathogen Anaplasma marginale establishes lifelong persistent infection in the mammalian reservoir host, during which time immune escape variants continually arise in part because of variation in the expressed copy of the immunodominant outer membrane protein MSP2. A key question is how the small 1.2 Mb A. marginale genome generates sufficient variants to allow long-term persistence in an immunocompetent reservoir host. The recombination of whole pseudogenes into the single msp2 expression site has been previously identified as one method of generating variants, but is inadequate to generate the number of variants required for persistent infection. In the present study, we demonstrate that recombination of a whole pseudogene is followed by a second level of variation in which small segments of pseudogenes recombine into the expression site by gene conversion. Evidence for four short sequential changes in the hypervariable region of msp2 coupled with the identification of nine pseudogenes from a single strain of A. marginale provides for a combinatorial number of possible expressed MSP2 variants sufficient for lifelong persistence.

Amino Acid Sequence↗

PubMed1985

Anaplasma marginale infections in American bison: experimental infection and serologic study.

Anaplasma marginale was experimentally transmitted from cattle to bison and back to cattle. Of the 2 splenectomized and 1 intact American bison calves (Bison bison) inoculated with a North Texas A marginale stabilate, 1 splenectomized and 1 intact bison exhibited clinical signs of anaplasmosis. Active parasitemias in these bison were observed along with positive reactions in the rapid card agglutination and complement fixation tests. Blood from the infected bison produced disease in splenectomized bovine calves. Screening tests for anti-Anaplasma antibodies in 178 blood samples collected from adult bison from the National Bison Range, Montana, revealed 1 rapid card agglutination test-positive sample, and 110 negative, 40 suspect, and 28 positive (15.7%) complement fixation test samples.

Agglutination Tests↗

PubMed1998

Morphology and development of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in cultured Ixodes scapularis (Acari: Ixodidae) cells.

Anaplasma marginale Theiler, a tick-borne rickettsial pathogen of cattle, was recently propagated in a continuous tick cell line, IDE8, derived from embryonic Ixodes scapularis Say. Cell monolayers were infected briefly with a high multiplicity of infection to synchronize rickettsial development and allow for description of the invasion, development, and release of A. marginale from the cultured cells. Sequential samples were collected, fixed, and processed for examination with light and electron microscopy. A. marginale entered host cells by an endocytotic process and remained within a vacuolar membrane throughout development. After entry, the dense form of A. marginale transformed into the vegetative or reticulated form that multiplied by binary fission, forming large colonies of rickettsiae. The reticulated form subsequently transformed into the dense form of A. marginale, which was released from cells and survived extracellularly. The dense forms were eventually released from the cultured cells by a process in which the inclusion membrane fused with the host cell membrane. Release of A. marginale was effected without the loss of host cell cytoplasm. In subsequent cell cycles, A. marginale reinfected cultured cells resulting in the development of multiple colonies per cell and eventual host cell destruction. Small vesicles were abundant within the colonies and appeared to form from individual rickettsiae. Development of A. marginale in IDE8 cells was similar to that described in naturally infected Dermacentor spp. ticks. However, destruction of cells by A. marginale as seen in vitro was not observed in naturally infected ticks. An understanding of the developmental cycle of A. marginale in cultured cells may provide insight into rickettsial development in its tick host and provide a basis for studying pathogen-host cell interaction in vitro.

Anaplasma↗

PubMed2001

Expression of Anaplasma marginale major surface protein 2 variants in persistently infected ticks.

Anaplasma marginale, an intraerythrocytic ehrlichial pathogen of cattle, establishes persistent infections in both vertebrate (cattle) and invertebrate (tick) hosts. The ability of A. marginale to persist in cattle has been shown to be due, in part, to major surface protein 2 (MSP2) variants which are hypothesized to emerge in response to the bovine immune response. MSP2 antigenic variation has not been studied in persistently infected ticks. In this study we analyzed MSP2 in A. marginale populations from the salivary glands of male Dermacentor variabilis persistently infected with A. marginale after feeding successively on one susceptible bovine and three sheep. New MSP2 variants appeared in each A. marginale population, and sequence alignment of the MSP2 variants revealed multiple amino acid substitutions, insertions, and deletions. These results suggest that selection pressure on MSP2 occurred in tick salivary glands independent of the bovine immune response.

Amino Acid Sequence↗

PubMed2004

Complete genome sequencing of Anaplasma marginale reveals that the surface is skewed to two superfamilies of outer membrane proteins.

The rickettsia Anaplasma marginale is the most prevalent tick-borne livestock pathogen worldwide and is a severe constraint to animal health. A. marginale establishes lifelong persistence in infected ruminants and these animals serve as a reservoir for ticks to acquire and transmit the pathogen. Within the mammalian host, A. marginale generates antigenic variants by changing a surface coat composed of numerous proteins. By sequencing and annotating the complete 1,197,687-bp genome of the St. Maries strain of A. marginale, we show that this surface coat is dominated by two families containing immunodominant proteins: the msp2 superfamily and the msp1 superfamily. Of the 949 annotated coding sequences, just 62 are predicted to be outer membrane proteins, and of these, 49 belong to one of these two superfamilies. The genome contains unusual functional pseudogenes that belong to the msp2 superfamily and play an integral role in surface coat antigenic variation, and are thus distinctly different from pseudogenes described as byproducts of reductive evolution in other Rickettsiales.

Anaplasma marginale↗

PubMed2004

Identification of a novel Anaplasma marginale appendage-associated protein that localizes with actin filaments during intraerythrocytic infection.

The rickettsial pathogen Anaplasma marginale assembles an actin filament bundle during intracellular infection. Unlike other bacterial pathogens that generate actin filament tails, A. marginale infects mature erythrocytes, and the F-actin appendages are assembled on the cytoplasmic surface of a vacuole containing several organisms. To identify A. marginale molecules associated with these filaments, two complementary approaches were used: matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and tandem mass spectrometry of A. marginale proteins identified with an appendage-specific monoclonal antibody and expression screening of an A. marginale phage library. Amino acid and nucleotide sequences were mapped to a full-length gene in the genome of the St. Maries strain of A. marginale; the correct identification was confirmed by expression of full-length recombinant protein and its reactivity with appendage-specific antibodies. Interestingly, there is marked variation in the abilities of diverse A. marginale strains to assemble the F-actin appendages. Comparison of four strains, the Florida, Illinois, St. Maries, and Virginia strains, revealed substantial polymorphism in the gene encoding the appendage-associated protein, with amino acid sequence identity of as low as 34% among strains. However, this variation does not underlie the differences in expression, as there is no specific polymorphism associated with loss of ability to assemble actin appendages. In contrast, the ability to assemble an actin filament bundle reflected dramatic strain-specific differences in the expression level of the appendage-associated protein. Understanding how this protein influences the cycle of invasion, replication, and egress in the host cell may provide new insights into pathogen-host interactions.

Actins↗

PubMed1991

Crossprotective immunity between the Florida and a Zimbabwe stock of Anaplasma marginale.

Cattle immunised by infection with the Florida stock of Anaplasma marginale were protected against subsequent homologous challenge, as demonstrated by complete prevention of microscopically detectable parasitaemia. Identically immunised cattle were partially protected against challenge with the Norton, Zimbabwe stock of A. marginale as determined by the significant prolongation of the prepatent period, a significantly lower peak parasitaemia, and a significantly lower percentage drop in haemoglobin concentration when compared to non-immunised calves challenged identically. The partial protection induced by live Florida stock immunisation demonstrates that while protection-inducing epitopes are shared between the two stocks, induction of complete immunity against a Zimbabwe stock may require alternative presentation of Florida stock epitopes or inclusion of additional Zimbabwe stock epitopes in the immunogen.

Anaplasma↗

PubMed1982

A case of apparent suppression of Anaplasma marginale infection by eperythrozoonosis (Eperythrozoon teganodes).

The apparent suppression of Anaplasma marginale infection by Eperythrozoon teganodes in a splenectomized calf has been reported. A splenectomized calf, inoculated with 500 ml of blood having 23% erythrocytes infected with A. marginale, developed eperythrozoonosis on the fourth day post inoculation. A. marginale parasitaemia remained very low during the patent eperythrozoonosis. A. marginale parasites started to increase in number only after E. teganodes infection had been controlled with neoarsphenamine. A splenectomized calf treated identically, but not showing E. teganodes parasites in the peripheral blood, developed clinical anaplasmosis and fulminant parasitaemia within 3-4 days post inoculation.

Anaplasmataceae Infections↗

PubMed1998

Expression of Anaplasma marginale major surface protein 2 variants during persistent cyclic rickettsemia.

Anaplasma marginale is an intraerythrocytic rickettsial pathogen of cattle in which infection persists for the life of the animal. Persistent A. marginale infection is characterized by repetitive rickettsemic cycles which we hypothesize reflect emergence of A. marginale antigenic variants. In this study, we determined whether variants of major surface protein 2 (MSP-2), a target of protective immunity encoded by a polymorphic multigene family, arise during persistent rickettsemia. By using a quantitative competitive PCR to identify rickettsemic cycles, msp-2 transcripts expressed in vivo were isolated from peak rickettsemia of sequential cycles. Cloning and sequencing of msp-2 cDNA revealed that genetic variants of MSP-2 emerge representing a minimum of four genetic variant types in each cycle during persistent infection. Two-color immunofluorescence using variant-specific antibody showed that emergence of MSP-2 variants resulted in expression of a minimum of three antigenic types of MSP-2 within one rickettsemic cycle. Therefore immune control of each cycle would require responses to an antigenically diverse A. marginale population. These findings demonstrate that polymorphic MSP-2 variants emerge during cyclic rickettsemia in persistent A. marginale infection and suggest that emergent variants play an important role in persistence.

Amino Acid Sequence↗

PubMed2005

Relative efficiency of biological transmission of Anaplasma marginale (Rickettsiales: Anaplasmataceae) by Dermacentor andersoni (Acari: Ixodidae) compared with mechanical transmission by Stomoxys calcitrans (Diptera: Muscidae).

Anaplasma marginale Theiler is a tick-borne intraerythrocytic rickettsial pathogen of cattle that also can be mechanically transmitted by biting flies. Rickettsemia during the acute phase of infection may reach as high as 10(9) infected erythrocytes (IEs) per milliliter of blood. Animals that survive acute infection develop a life-long persistent infection that cycles between 10(2.5) and 10(7) IE/ ml of blood. We compared stable fly Stomoxys calcitrans (L.) -borne mechanical transmission during acute infection with Rocky Mountain wood tick, Dermacentor andersoni Stiles-borne biological transmission in the persistent phase of infection to demonstrate quantitatively that biological transmission by ticks is considerably more efficient than mechanical transmission by biting flies. Stable flies that partially fed on an acutely infected calf and were immediately transferred to susceptible calves to complete their bloodmeals failed to transmit A. marginale. Ticks that fed on the original acquisition host after it reached the persistent phase of infection (>300-fold lower rickettsemia) successfully transmitted A. marginale after transfer to the same calves that failed to acquire infection after fly feeding. Failure of fly-borne mechanical transmission at a rickettsemia >300-fold higher than that from which ticks transmit with 100% efficiency demonstrates that tick-borne biological transmission is at least 2 orders of magnitude more efficient than direct stable fly-borne mechanical transmission.

Anaplasma marginale↗

PubMed1986

Percutaneous infection of nymphal Dermacentor andersoni with Anaplasma marginale.

Newly replete nymphal Dermacentor andersoni (principals) were percutaneously exposed to Anaplasma marginale by injection of either intact or lysed infected bovine erythrocytes. Control nymphs were fed on calves with anaplasmosis. The subsequently molted adults were examined for infection by light microscopy, and companion ticks were tested for infectivity by allowing them to feed on susceptible calves. When they fed as adults, both control ticks and percutaneously inoculated principals transmitted A marginale to susceptible calves. Prepatent periods in calves varied according to the method by which nymphs were infected. Colonies of A marginale were found in all ticks that acquired infection by feeding, but colonies were not observed in any ticks exposed percutaneously. The possible developmental cycle of A marginale in artificially infected ticks is discussed.

Anaplasma↗

PubMed2006

Development of enzyme-linked immunosorbent assays based on recombinant MSP1a and MSP2 of Anaplasma marginale.

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant MSP1a and MSP2 from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. The high sensitivities (99% for both tests) and specificities (100% for both tests) were confirmed with sera from cattle positive or negative for A. marginale antibodies, respectively, by immunofluorescent antibody test. By the analysis of 583 sera from cattle of three regions of the state of Pernambuco, Brazil, the agreement between both tests was high, with a kappa index of 0.89. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Anaplasma marginale↗

PubMed1976

Survival of Anaplasma marginale in Aedes albopictus cells.

Bovine red blood cells infected with Anaplasma marginale were introduced into cell line cultures from the mosquito Aedes albopictus. The cultured mosquito cells phagocytized both infected and uninfected red blood cells. The parasite remained viable, as determined by calf inoculations, for 21 days.

Aedes↗