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PubMed2006

A Canadian bison isolate of Anaplasma marginale (Rickettsiales: Anaplasmataceae) is not transmissible by Dermacentor andersoni (Acari: Ixodidae), whereas ticks from two Canadian D. andersoni populations are competent vectors of a U.S. strain.

Anaplasma marginale Theiler is a tick-borne rickettsial pathogen of cattle with a global distribution in both temperate and tropical regions. The pathogen is endemic in regions within the United States, whereas the Canadian cattle population is considered to be free ofA. marginale. Farmed bison, Bison bison L., in central Saskatchewan have been found to be infected with A. marginale; however, there is no evidence of transmission from bison to cattle. We tested a Saskatchewan bison isolate of A. marginale (SB1) to determine whether it is transmissible by the Rocky Mountain wood tick, Dermacentor andersoni Stiles. Colonized D. andersoni from the United States and Canada failed to transmit SB1. A separate transmission trial using D. andersoni adults reared from ticks collected in Alberta and British Columbia showed that ticks from these populations could successfully transmit the St. Maries, Idaho, strain of A. marginale. Although the Saskatchewan bison isolate of A. marginale seems not to be transmissible by D. andersoni, in the event of the introduction of a tick-transmissible strain, Canadian D. andersoni are likely to be competent vectors.

Anaplasma marginale↗
PubMed1997

In vitro cultivation of Anaplasma marginale in bovine erythrocytes co-cultured with endothelial cells.

Primary cultures of Anaplasma marginale infected erythrocytes were used to determine conditions for in vitro cultivation of the rickettsia. The infected erythrocytes that were maintained by regular addition of Glasgow's MEM with fetal calf serum and uninfected erythrocytes showed a 1-5% increase in percent infected erythrocytes on the evaluation of Giemsa stained smears. This increase in parasitemia resulted in up to 70% change in the number of infected erythrocytes. Co-culture of the infected erythrocytes with endothelial cell monolayers allowed for longer maintenance with the parasitemia ranging from 5-13% through four passages over 16 weeks. Examination of cultures using transmission electron microscopy showed initial bodies within the erythrocytes at 10 days after the initial passage of the primary culture. The endothelial cell monolayers in the co-cultures contained multiple initial bodies. We have demonstrated that A. marginale can be grown for a limited number of passages in the co-culture system, which will facilitate the development of a continuous culture of the organism.

Anaplasma↗
PubMed1998

Conformational dependence of Anaplasma marginale major surface protein 5 surface-exposed B-cell epitopes.

The Anaplasma marginale outer membrane is composed of immunogenic major surface proteins (MSPs) linked both covalently and noncovalently in multimeric complexes (M. C. Vidotto, T. C. McGuire, T. F. McElwain, G. H. Palmer, and D. P. Knowles, Infect. Immun. 62:2940-2946). Consequently, effective induction of antibody against surface-exposed MSP epitopes has been postulated to require maintenance of MSP secondary through quatenary structures. Using MSP5 as a model and the approach of epitope mapping with recombinant expressed full-length and truncated proteins, we demonstrated that the immunodominant surface epitope bound by monoclonal antibody (MAb) ANAF16C1 required disparate amino- and carboxy-terminal regions of MSP5, indicating the conformational dependence of this epitope. The required amino-terminal MSP5 region included the cysteines involved in intramolecular disulfide bonding. The dependence of the immunodominant epitope on disulfide bonding was confirmed by loss of MAb ANAF16C1 binding to MSP5 following disulfide bond reduction and covalent modification of the reduced sulfhydryl groups. The recognition of the MSP5 immunodominant epitope by antibody induced by protective immunization with A. marginale outer membranes was also conformationally dependent, as shown by the loss of epitope binding following serum adsorption with native but not reduced and denatured A. marginale. Importantly, the antibody response to all immunodominant MSP5 surface epitopes was restricted to conformationally dependent epitopes, since the binding of polyclonal anti-MSP5 antibody to the A. marginale surface could be blocked by adsorption with native but not denatured and reduced MSP5. These results confirm the importance of the secondary and tertiary structures of MSP epitopes as immune system targets and support the testing of immunogens which maintain the required conformation.

Anaplasma↗
PubMed2006

Comparison of the efficacy of enrofloxacin, imidocarb, and oxytetracycline for clearance of persistent Anaplasma marginale infections in cattle.

This study compared enrofloxacin and imidocarb dipropionate treatments with an oxytetracycline regimen proposed by the World Organization for Animal Health for elimination of persistent Anaplasma marginale infections in cattle. The effect of therapy on competitive ELISA and polymerase chain reaction (PCR) reactivity was also assessed. Twelve A. marginale-infected carrier calves were randomly assigned to groups receiving either enrofloxacin (5 mg/kg IV q24h for 5 days), imidocarb (5 mg/kg IM twice, 7 days apart), or oxytetracycline (22 mg/kg IV q24h for 5 days). One calf infected with an Oklahoma isolate in the imidocarb group and one infected with a Virginia isolate in the oxytetracycline group failed to infect a splenectomized calf following blood subinoculation. Both became competitive ELISA negative by 44 days after treatment, but the imidocarb-treated calf remained PCR positive. None of the tested treatments reliably eliminated persistent A. marginale infections in all cattle. Furthermore, PCR was not a reliable means of determining the success of chemosterilization in calves.

Anaplasma marginale↗
PubMed1988

Differential extraction of antigens of Anaplasma marginale.

Strain-, group-, and genus-specific antigens of the Florida isolate of Anaplasma marginale had different solubilities in zwitterionic, nonionic, and anionic detergents. On the basis of their solubility in nonionic detergent, antigens were grouped into 3 classes: (i) a 108-kilodalton (kD) group-specific antigen and a series of poorly defined antigens (70 to 100 kD) were completely soluble in nonionic detergent; (ii) 96 kD and 75 kD group-specific antigens, and a 91-kD strain-specific antigen were completely insoluble in nonionic detergent; and (iii) a 47-kD group-specific antigen and a 41-kD genus-specific antigen were partially soluble in nonionic detergent. A mercaptoethanol-sensitive antigen of 200 kD also was present in the insoluble fraction. Differences in solubility of taxonomic antigens of Anaplasma may reflect differences in function. Antigens totally insoluble in nonionic detergent were possibly integral membrane proteins, and those completely soluble in nonionic detergent were more likely peripheral membrane proteins that were less tightly bound to hydrophobic components of the membrane. Seemingly, all antigens were associated with membranes. Bovine preimmune and immune sera used did not react with any proteins of noninfected RBC.

Anaplasma↗
PubMed1985

Detection of geographic isolates of Anaplasma marginale, using polyclonal bovine antisera and microfluorometry.

Geographically separate United States isolates of Anaplasma marginale were differentiated, using polyclonal bovine antiserum and microfluorometry. Both isolate-common and restricted antigen-specific antibodies were apparent in sera of splenectomized calves after resolution of infection, as judged by the capability of the antisera to recognize heterologous isolate antigens to a lesser extent than homologous antigens. Furthermore, absorption of the antisera with homologous antigens removed homologous and heterologous reactions, whereas heterologous absorptions resulted in the capability to discriminate the tailed or appendage-associated isolates (Virginia and Washington) from the tail-less isolate (Florida).

Anaplasma↗
PubMed1979

In vitro cultivation of Anaplasma marginale: invasion of and development in noninfected erythrocytes.

Ovine erythrocytes infected with attenuated Anaplasma marginale organisms were cultured in a suspension of normal ovine erythrocytes and normal bovine erythrocytes for 42 days. In each system, the organism showed an initial period of rapid growth followed by a gradual decrease in the percentage of parasitized erythrocytes accompanied by cyclic peaks. The percentage of infection of ovine erythrocytes were not different when normal ovine or bovine erythrocytes were added to the cultures. In vitro transmission of the organism from infected ovine cells to normal bovine cells was demonstrated by use of a two-step direct fluorescent antibody method, which allowed for specific identification of the two cell types and the organism.

Anaplasma↗
PubMed1977

Resistance to anaplasmosis after elimination of latent Anaplasma marginale infections.

Ten 30-month-old cattle (group 2) were inoculated with Anaplasma marginale 6 months after the latent A marginale infection was eliminated by oral chlortetracycline therapy. In addition, 4 cattle (group 1) with no record of infection and 4 cattle (group 3) of equivalent age previously vaccinated with a killed antigen were similarly inoculated. Moderate to severe clinical signs of acute anaplasmosis occurred in the group 1 (previously uninfected) cattle, whereas only 1 of the chlortetracycline-treated cattle (group 2) and none of the vaccinated cattle (group 3) had clinical evidence of disease.

Anaplasmosis↗
PubMed2004

Mapping of B-cell epitopes in the N-terminal repeated peptides of Anaplasma marginale major surface protein 1a and characterization of the humoral immune response of cattle immunized with recombinant and whole organism antigens.

Major surface protein (MSP) 1a of the genus type species Anaplasma marginale (Rickettsiales: Anaplasmataceae) together with MSP1b forms the MSP1 complex. MSP1a has been shown to be involved in adhesion, infection and tick transmission of A. marginale, as well as to contribute to protective immunity in cattle. A differential antibody response to MSP1a and MSP1b was observed in cattle immunized with A. marginale derived from bovine erythrocytes (anti-MSP1a response) or cultured tick cells (anti-MSP1b response). In this study, we further characterized the MSP1a antibody response of cattle using several immunogens, including recombinant MSP1a (rMSP1a) protein, erythrocyte- or tick cell culture-derived A. marginale, or a combination of tick cell culture-derived A. marginale and rMSP1a. The MSP1a antibody response to all these immunogens was directed primarily against the N-terminal region of MSP1a that contains tandemly repeated peptides, whereas low antibody levels were detected against the C-terminal portion. Linear B-cell epitopes of MSP1a were mapped using synthetic peptides representing the entire sequence of the protein that were prepared by SPOT synthesis technology. Only two peptides in the N-terminal repeats were recognized by sera from immunized cattle. These peptides shared the sequence SSAGGQQQESS, which is likely to contain the linear B-cell epitope that was recognized by the pools of bovine sera. The average differential of antibody titers against MSP1a minus those against MSP1b correlated with lower percent reductions in PCV. A preferential antibody response to MSP1a was observed in cattle immunized with erythrocyte-derived, cell culture-derived plus rMSP1a or rMSP1a alone, and the percent reduction PCV was significantly lower in these cattle as compared with the other immunization groups. These results provide insight into the bovine antibody response against A. marginale and the role of MSP1a in protection of cattle against A. marginale infection.

Anaplasma marginale↗
PubMed2001

Sensitivity and specificity of the complement fixation test for detection of cattle persistently infected with Anaplasma marginale.

The complement fixation (CF) test commonly is used to identify cattle infected with Anaplasma marginale prior to interstate or international movement. Estimates of the accuracy of the CF test in detecting animals persistently infected with A. marginale vary widely. In this study, the sensitivity and specificity of the CF test for detection of carrier animals was determined using serum from 232 cattle previously defined as A. marginale positive or negative by nested polymerase chain reaction methods and hybridization. Considering results from 2 independent laboratories and interpreting a 1:5 suspect reaction as positive, the best estimate of CF test sensitivity was 20%, with a specificity of 98%. Using a 1:10 cutoff, sensitivity decreased to 14% and specificity increased to 99%. Results of this study indicate that the CF test is ineffective for identifying cattle persistently infected with A. marginale and thus is inadequate for anaplasmosis regulatory and surveillance programs.

Anaplasma↗
PubMed1996

Morphology and development of Anaplasma marginale in midgut of engorged female ticks of Boophilus microplus.

Morphology and development of Anaplasma marginale were studied in midgut epithelial cells of adult Boophilus microplus females using light and transmission electron microscopy. Hemoparasite-free B. microplus larvae were used to infest Holstein calves experimentally inoculated with A. marginale. When A. marginale parasitemia varied from 0.4% to 4.3%, 140 engorged female ticks were collected and kept individually in steel wire tubes and incubated outdoors on Brachiaria decumbens grass pasture. Female ticks in groups of 20 were dissected 14, 19, 26, 33, 40, 47 and 50 days after detachment. Engorged female ticks collected from an A. marginale-free calf were kept under the same conditions as control ticks. Gut sections of infected and control ticks were processed using light and electron microscopy. Infected gut sections were processed by the immunocytochemical peroxidase-antiperoxidase technique. Under light microscopy A. marginale colonies were found in midgut epithelial cells of engorged B. microplus females from 19 days on after detachment from donor calves. Colonies were 6.1-10.3 microns in diameter, ovoid or irregular and contained round or shaped organisms. Two distinct morphological types of colonies (BM1 and BM2) were found. BM1 colonies had a dense matrix strongly stained in which organisms were barely visible; BM2 had large separated organisms densely stained inside a matrix which was lightly stained. A. marginale colonies immunocytochemically stained were found in gut tissue sections using light microscopy. Using transmission electron microscopy organisms of different morphologies were found in midgut epithelial cells of engorged females, suggesting sequential phases of development of A. marginale in the vector. Modifications occurred in colonies and in the organisms within them. No symbiotic rickettsiae were found in intestinal cells of control ticks.

Anaplasma↗
PubMed1983

Comparison of proteins synthesized by two different isolates of Anaplasma marginale.

We present results on the initial definition of proteins synthesized by two isolates of Anaplasma marginale. Bovine erythrocytes infected with A. marginale were radioactively labeled with [35S]methionine or a 3H-amino acid mixture during short-term in vitro culture. The labeled proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This technique revealed protein bands of various apparent molecular weights from less than 14,000 to greater than 200,000. The bands observed represented A. marginale proteins because (i) uninfected erythrocytes from the same animal did not incorporate radioisotope during identical culture conditions, and (ii) the incorporation of radioisotope into proteins during culture of infected erythrocytes was inhibited by tetracycline but not by cycloheximide. The radioactive protein profiles of two different isolates of A. marginale, from Washington and Florida, were compared by two-dimensional gel electrophoresis. About 200 proteins were resolved in each case. Several proteins differed in position when the two-dimensional gel maps were compared, indicating variations in protein structure between the two A. marginale isolates.

Anaplasma↗
PubMed2005

Anaplasma marginale: lack of cross-protection between strains that share MSP1a variable region and MSP4.

In Mexico, there are no commercial alternatives for the immunoprophylaxis of bovine Anaplasmosis, a disease responsible for great economic losses. Blood derived Anaplasma marginale used for immunizing susceptible cattle has shown promising results for homologous protection and controversial results against unrelated strains. The present study examined, under controlled conditions, the cross-protective potential of an immunogen composed of blood derived A. marginale of three strains against challenge with strains not included in the immunogens. Groups 1 and 2 were immunized with blood derived Anaplasma from strains Mexico, Morelos and Yucatan, group 4 with strains Morelos, Veracruz and Yucatan, two more groups (2 and 5) of equal conditions were inoculated with an adjuvant alone. Groups 1, 4 and 5 were challenged with Mexico strain; groups 2 and 3 were challenge-inoculated with strain Veracruz; groups 3 and 5 with strains Veracruz and Mexico as controls. Only animals in group 1, immunized and challenged with strain Mexico showed adequate protection. Both groups challenged with strains not included in the immunogens developed poor protection, while all the controls had to be treated to prevent death.

Anaplasma marginale↗
PubMed2002

Applications of a cell culture system for studying the interaction of Anaplasma marginale with tick cells.

A cell culture system for the tick-borne rickettsia Anaplasma marginale offers new opportunities for research on this economically important pathogen of cattle. A. marginale multiplies in membrane-bound inclusions in host cells. Whereas erythrocytes appear to be the only site of infection in cattle, A. marginale undergoes a complex developmental cycle in ticks and transmission occurs via the salivary glands during feeding. We recently developed a cell culture system for A. marginale using a cell line derived from embryos of Ixodes scapularis. Here we review the use of this cell culture system for studying the interaction of A. marginale with tick cells. Several assays were developed using the A. marginale/tick cell system. An adhesion assay was developed for the identification of proteins required by A. marginale for adhesion to tick cells. The effect of antibodies against selected major surface proteins in inhibiting A. marginale infection was tested in an assay that allowed further confirmation of the role of surface proteins in the infection of tick cells. A drug screening assay for A. marginale was developed and provides a method of initial drug selection without the use of cattle. The culture system was used to test for enhancing effects of tick saliva and saliva components on A. marginale infection. The tick cell culture system has proved to be a good model for studying A. marginale-tick interactions. Information gained from these studies may be applicable to other closely related tick-borne pathogens that have been propagated in the same tick cell line.

Anaplasma↗
PubMed1986

Isolate of Anaplasma marginale not transmitted by ticks.

The tick-borne transmissibility of 2 isolates of Anaplasma marginale was compared. Dermacentor variabilis were exposed to A marginale as nymphs by feeding on 1 of 4 splenectomized calves during periods of ascending parasitemia (maximum 49% to 81% parasitized erythrocytes) induced by injection of a stabilate. Tick-borne transmission was attempted, using 26 to 224 adult ticks within 30 to 220 days after molting. Adult D variabilis did not transmit an Illinois isolate of A marginale in 7 tick-borne transmission experiments (P = 0.0047), including 2 experiments in which calves were inoculated IV with homogenates of adult ticks. In contrast, a Virginia isolate of A marginale was readily transmitted by the same tick colony. Thus, previously reported morphologic and immunologic differences among A marginale isolates may extend to tick-borne transmissibility. The Virginia and Illinois A marginale isolates had an inclusion appendage that was not a marker for tick transmissibility.

Anaplasmosis↗
PubMed2004

Prevalence of antibodies to bluetongue virus and Anaplasma marginale in Montana yearling cattle entering Alberta feedlots: Fall 2001.

A serologic survey was conducted in yearling cattle imported into Alberta feedlots from Montana during October 2001 to estimate the prevalence of antibodies to bluetongue virus (BTV) and Anaplasma marginale in Montana yearling cattle. The apparent prevalence of antibodies to BTV when the competitive enzyme-linked immunosorbent assay (cELISA) was used was 0.37% (21/5608). Test positive cELISA samples were also all positive when tested by virus neutralization (VN) and they reacted to 1 or more BTV serotypes, including 2, 10, 11, 13, and 17. The apparent prevalence of antibodies to A. marginale when a recombinant cELISA (rcELISA) was used with a positive cutoff at 30% inhibition was 1.93% (108/5608). When the rcELISA positive cutoff was at 42% inhibition, the apparent prevalence was 0.73% (41/5608). After the reported sensitivity and specificity of the test had been accounted for, the A. marginale antibody results were consistent with a population that was either free of exposure or had a very low prevalence for A. marginale.

Alberta↗
PubMed1998

Development of a non-radioactive DNA probe and in situ hybridization for detection of Anaplasma marginale in ticks and cattle.

A non-radioactive DNA probe was developed for detection of Anaplasma marginale in ticks and cattle. The probe was labeled with digoxigenin 11-dUTP by polymerase chain reaction. The probe was tested on bovine blood and was found to be a sensitive and specific detection method for A. marginale in cattle. The DNA probe was then adapted for in situ hybridization (ISH) of A. marginale in Dermacentor andersoni and D. variabilis ticks infected either as nymphs or adults. One-half of each tick was studied with ISH while the other half was examined with light and electron microscopy. In male ticks infected as adults, tick gut cells first became infected with A. marginale while ticks fed on an infected calf, and they remained infected as they transmission fed on a second, susceptible calf. At the onset of transmission feeding, salivary glands became infected with A. marginale. During transmission feeding infection was also observed in interstitial, reproductive, skeletal muscle, fat body and Malpighian tubule tissue, resulting in a generalized A. marginale infection. When adult ticks that acquired infection as nymphs were examined with ISH and microscopy, gut tissues of both D. andersoni and D. variabilis became infected with A. marginale. However, salivary gland infection was seen only in D. variabilis, even though both species of ticks transmitted A. marginale to susceptible calves. A. marginale was not seen with ISH or microscopy in hemocytes collected from both species of ticks and, thus, hemocytes do not appear to play a role in the development of A. marginale in ticks.

Anaplasma↗