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PubMed2004

Identification of IgG2-specific antigens in Mexican Anaplasma marginale strains.

To identify novel antigens with immunoglobulin G2 (IgG2) specificity and immunostimulant properties for bovine Th1 cells, humoral and cellular responses were studied in cattle inoculated with initial bodies from a Mexican isolate of Anaplasma marginale and challenged with a heterologous strain. Analysis of post-immunization sera by ELISA and assaying of in vitro cellular responses in peripheral blood mononuclear cells (PBMCs) cultured in the presence of protein extracts from three Anaplasma marginale strains showed positive values of optical density ELISA readings and stimulation indices in the immunized but not control cattle. Post-immunization and post-challenge sera recognized in Western blots several proteins with molecular weights ranging from 15 to 209 kDa, twelve of which were recognized by IgG2 in the three Anaplasma marginale strains. Seven of these are novel and have not been previously reported for their IgG2 specificity; three are confirmed to be major surface proteins (MSP-1a, MSP-2, and MSP-5); and the others correspond to other well-studied MSPs but were not confirmed. Partially purified fractions of protein extracts of the Mex-17 strain were tested against PBMCs cultured in vitro. One out of the seven novel proteins induced detectable lymphoproliferation (LP) of PBMCs, and interferon-gamma was detected in supernatants of PBMC cultured in the presence of two protein fractions, including the one that caused LP. It is concluded that novel antigens, particularly the 28-kDa protein, played an additional role in the protection of immunized cattle and should be considered vaccine candidates after in vivo immunization experiments are concluded.

Anaplasma marginale↗
PubMed1993

A retrospective serologic survey for Anaplasma spp. infection in three bighorn sheep (Ovis canadensis) populations in California.

Using an indirect immunofluorescence assay, we determined the prevalence of Anaplasma-reactive antibody in three herds of bighorn sheep, each a different subspecies and occupying a different habitat in California (USA). Antibodies to Anaplasma spp. were identified in none of twenty California bighorn (Ovis canadensis californiana) sampled from the Mt. Baxter herd, 11 of 17 peninsular bighorn (O. canadensis cremnobates) sampled in the Santa Rosa Mountains, and all 20 desert bighorn (O. canadensis nelsoni) sampled at Old Dad Peak/Kelso Mountains. Based on an assay and an adsorption technique, the titers most likely were due to Anaplasma ovis. The presence and species of tick vectors in each of the habitats, and the presence or absence of deer or livestock were identified as factors potentially influencing seroprevalence of antibodies.

Adsorption↗
PubMed1996

Susceptibility of elk (Cervus elaphus) to experimental infection with Anaplasma marginale and A. ovis.

Anaplasma ovis was experimentally transmitted from domestic sheep to elk (Cervus elaphus) and back to splenectomized sheep. No rickettsemias were detected but serum from three of seven experimentally inoculated elk developed Anaplasma spp.-reactive antibody as measured by indirect immunofluorescence (IIF) or by the rapid card agglutination and complement fixation assays. Three elk were experimentally infected with A. marginale. The rickettsiae were detected in blood of these elk and caused disease in a splenectomized domestic bovine calf after subinoculation of blood from the elk. All three elk had positive titers with IIF. No clinical signs of illness were noted in any elk inoculated with either Anaplasma species.

Anaplasma↗
PubMed1975

Ultrastructure of Anaplasma marginale after freeze-fracture.

Stained thin sections and freeze-fractured replicas of Anaplasma marginale-infected bovine erythrocytes were examined by electron microscopy. Freeze-fracture replication not only verified basic Anaplasma ultrastructure, but also allowed visualization of structures not previously reported. Because of the partial 3-dimensional views obtained with freeze-fracture replication, a new structure that appears as a protruding tip was discernible. Also, the surface of Anaplasma's limiting membrane was less granular than the fractured surface of host erythrocyte. A corrugated surface with a periodicity of 10.5 nm was seen when the limiting membrane was fractured.

Anaplasma↗
PubMed2003

First evidence of Anaplasma platys in Rhipicephalus sanguineus (Acari: Ixodida) collected from dogs in Africa.

A total of 27 ticks, comprising Rhipicephalus sanguineus (Latreille) (n = 21), Haemaphysalis leachi (Andouin) (n = 4) and Haemaphysalis paraleachi (Camicas, Hoogstraal & El Kammah) (n = 2) were recovered from two clinically healthy female dogs in the Democratic Republic of the Congo. DNA of Anaplasma platys was detected in a female R. sanguineus, using primers derived from the 16S rRNA gene, which amplify members of the family Anaplasmataceae . Anaplasma platys DNA was also detected in the blood of one of the dogs. Phylogenetic analysis based on partial sequences of the 16S rRNA, the gltA and the groEL genes ranged the detected agent within the Anaplasma clade. This is the first reported detection of A. platys in ticks in Africa. This finding raises the question of the possible involvement of R. sanguineus in A. platys infection of dogs.

Amino Acid Sequence↗
PubMed2004

Comparing the sensitivity of anaplasma phagocytophilum DNA detection in Ixodes ricinus ticks by amplifying a fragment of the epank-1 and the 16s rDNA genes.

The study compared the sensitivity of two genetic markers (epank1, 16S rDNA) in identifying anaplasma DNA by using the polymerase chain reaction (PCR) technique. Material for anaplasma DNA studies was isolated from I. ricinus. Amplification of the 16S rDNA gene resulted in 5.77% positive samples, the value for the epank-1 gene equaled 11.54%. The results suggest that the sensitivity of the reaction for the epank-1 gene is greater than that of 16S rDNA for the identification of anaplasma DNA from isolates of I. ricinus. The application of epank- in identifying A. phagocytophilum DNA can change the pattern of infection in these arachnids in a given area. The results obtained in this study and cared out by another authors point at small useful a conservative gene as a marker for diagnostic goals.

Anaplasma phagocytophilum↗
PubMed2004

[Anaplasma phagocytophila and protozoans of Babesia genus in dogs from endemic areas of Lyme disease in north-western Poland].

Infections caused by the spirochete Borrelia burgdorferi sensu lato may be accompanied by other microorganisms, such as Anaplasma, Ehrlichia and Babesia. These pathogens are transmitted by the ticks and are a risk to humans and animals. Ixodes ricinus ticks collected from recreational areas of Szczecin and northwestern Poland contained DNA of the pathogens mentioned above and cases of double and triple coinfection have been documented. The aim of this paper was to determine if dogs suspect to tick infestation in the area of study are a reservoir for these pathogens and to examine the possibility of coinfection. Canine blood was sampled, part of the material originated from dogs exhibiting symptoms of borreliosis. In an earlier study, the samples were screened for DNA from B. burgdorferi sensu lato. In order to screen for A. phagocytophila and Babesia sp. DNA, a PCR-based method was used with the following primers: EHR521/EHR747 for Anaplasma and FOR1/REV1 for Babesia. In 192 samples only two contained A. phagocytophila DNA. One of these samples originated from a healthy canine, the other from an individual with symptoms of borreliosis. The examined samples were not positive for Babesia sp. DNA. Coinfection was not discovered. The low level of A. phagocytophila infection may indicate that the domestic dog is not a reservoir for Anaplasma and Babesia in Szczecin and northwestern Poland. Moreover, this area does not have populations of the brown dog tick (Rhipicephalus sanguineus) or Dermacentor reticulates--both of which are vectors of E. canis and B. canis and commonly induce ehrlichiosis and babesiosis in canines.

Anaplasma phagocytophilum↗
PubMed1990

Development and infectivity of Anaplasma marginale in Dermacentor andersoni nymphs.

The development of Anaplasma marginale was studied in Dermacentor andersoni nymphs after they had fed on a calf with ascending Anaplasma infection. Gut tissues were collected on day 4 of tick feeding, from newly replete (fed) nymphs and on postfeeding days (PFD) 5, 10, 15, 20, and were processed for light and electron microscopy to determine density of A marginale colonies. Homogenates of gut tissues were prepared from nymphs collected on the same days and inoculated into susceptible, splenectomized calves to test for infectivity. Anaplasma colonies were detected in gut cells on PFD 5, 10, 15, and 20. Although colony density appeared to be higher on PFD 10 and 15, differences were not significant. Nymphal type-1 colonies were detected in highest numbers on PFD 5 and 10, transitional colonies were seen in highest numbers at PFD 10 and 15, and nymphal type-2 colonies were observed only on PFD 20. Gut homogenates that were collected from ticks at 4 days of feeding, when newly replete, and on PFD 20 caused anaplasmosis when injected into susceptible calves, but homogenates made from ticks collected on PFD 5, 10, and 15 were not infective. The data indicate that of the colony types of A marginale that develop in replete nymphs, nymphal type-1 and transitional colonies may contain organisms that are not infective for cattle.

Anaplasma↗
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↗
PubMed1979

Electron microscope studies of Anaplasma marginale in an Aedes albopictus culture system.

Anaplasma marginale was seen by electron microscopy within the cytoplasm of Aedes albopictus cells in culture. Anaplasma marginale was seen in free bovine RBC as well as in RBC phagocytized by Ae albopictus cells. Some A marginale organisms, both intracellular and extracellular, remained intact for as long as 60 days in culture, and Anaplasma inclusion appendages were seen in free-lysed RBC and in lysed RBC phagocytized by Ae albopictus cells. Although some A marginale organisms had deteriorated by day 60, the structure of most A marginale organisms did not change appreciably from that in its natural bovine host. Reproduction of A marginale was not seen.

Aedes↗
PubMed1980

Ultrastructure of ANaplasma marginale Theiler in Dermacentor andersoni Stiles and Dermacentor variabilis (Say).

Cattle were inoculated with a Virginia isolate of Anaplasma marginale Theiler and served as an infective source for laboratory-reared Dermacentor andersoni Stiles and D variabilis (Say) nymphs. Anaplasma marginale was demonstrated by electron microscopy in gut tissues of replete nymphal ticks and in unfed, incubated, and feeding adult ticks that were exposed to the organism as nymphs when they fed on an infected cow. The A marginale organism in replete nymphs and adult feeding ticks were morphologically similar to A marginale described previously from infected bovine erythrocytes. Colonies of A marginale were demonstrated by light and electron microscopy in midgut epithelial cells of unfed adult D andersoni and D variabilis that had been incubated at 37 C for 3 days. Anaplasma marginale organisms in colonies were very pleomorphic. Small electron-dense particles were demonstrated in all infected tick stages studied, but were most evident in colonies from incubated, unfed ticks. They may represent a reproductive form of A marginale.

Anaplasma↗
PubMed1981

Comparative titration of Anaplasma marginale antibodies by card agglutination and complement-fixation tests.

The card agglutination test for anaplasmosis was improved so that bovine serum could be titrated for Anaplasma antibodies. The modified test was validated when its results on 1 set of serum samples were compared with the results furnished by the complement-fixation test on the same set. The samples tested were from cows inoculated with an Anaplasma vaccine or with either 1 of 2 virulent strains of Anaplasma marginale. A correlation coefficient of 0.60 was calculated between the values of the titers furnished by the 2 tests, and regression equations were calculated and used to estimate the serum titer in 1 test from a titer measured in the other. The results indicate that the simple card agglutination test can yield titers comparable with those of the complement-fixation test.

Agglutination Tests↗
PubMed1993

Persistence of tick-derived Anaplasma marginale in cultured bovine turbinate and endothelial cells.

Anaplasma marginale from salivary glands of Dermacentor andersoni was used to inoculate monolayers of bovine turbinate and endothelial cells. Monolayers were passaged at 2 or 4 week intervals and monitored with light and electron microscopy and with an A. marginale-specific DNA probe. Intracellular inclusions were observed in turbinate cells after 2-4 weeks. The number of inclusion-bearing cells increased over 1-2 weeks and gradually disappeared. A radiolabeled fragment from within the msp1 beta gene of the erythrocytic stage of A. marginale hybridized to DNA extracted from bovine turbinate cell cultures as late as 7 weeks post inoculation (passage 4). Individual rickettsiae were observed with electron microscopy in samples taken at this time. Susceptible calves inoculated with suspect cultures did not develop clinical anaplasmosis but did develop significant antibody titers as detected with ELISA. DNA from endothelial cell cultures at 9 weeks post inoculation also bound the Anaplasma-specific DNA probe. Anaplasma marginale from salivary glands of D. andersoni appears to persist in cultured bovine turbinate and endothelial cells but typical development and infectivity for bovines do not occur.

Anaplasma↗
PubMed1977

Cell-mediated immunity related to challenge exposure of cattle inoculated with virulent and attenuated strains of Anaplasma marginale.

Cattle which were given virulent Anaplasma marginale as a premunization procedure or were vaccinated with live attenuated or killed anaplasma preparations were challenge inoculated with virulent A marginale. Exposure of cattle to virulent or attenuated A marginale, which induced a strong and lasting cell-mediated response as measured by the leukocyte migration-inhibition test, protected against the development of high parasitemia, anemia, and clinical signs of disease after challenge inoculation with virulent A marginale. Cattle which were vaccinated with killed anaplasma preparations that induced transient and low-level cell-mediated responses were not protected against the development of severe anemia after challenge inoculation, although postchallenge parasitemia seemed to be controlled.

Anaplasma↗
PubMed1981

Recrudescence of Anaplasma marginale induced by immunosuppression with cyclophosphamide.

Eight doses of cyclophosphamide (5 mg/kg), at 2-day intervals between doses, were administered IV to Anaplasma-carrier splenectomized calves. Significant decreases in serum immunoglobulins, diminished complement-fixing antibody response, and transitory leukopenia were associated with the treatment. Recrudescence of clinical Anaplasma infection occurred after the 5th dose of cyclophosphate was given and was characterized by rapid increase in parasitemia, marked decrease in PCV, and mortality. The results of the present study indicated that humoral mediated immunity may contribute to the maintenance of a state of equilibrium in the host-parasite relationship and that suppression of humoral immune responses may alter the course and outcome of infection in Anaplasma carriers.

Anaplasmosis↗
PubMed2006

Reduction of tick infections with Anaplasma marginale and A. phagocytophilum by targeting the tick protective antigen subolesin.

Subolesin was recently shown by both gene silencing and immunization with the recombinant protein to protect against tick infestations, and to cause reduced tick survival and degeneration of gut and salivary gland tissues. In this research, we extended these studies by testing whether targeting subolesin by RNAi or vaccination interfered with the ability of ticks to become infected with two tick-borne pathogens, Anaplasma marginale which causes bovine anaplasmosis and Anaplasma phagocytophilum, the causative agent of human granulocytin anaplasmosis. For the A. marginale studies, Dermacentor variabilis males were injected with subolesin dsRNA or saline and then were allowed to feed on cattle with ascending rickettsemias, while for the A. phagocytophilum studies, mice were immunized with the recombinant subolesin protein, infected with the pathogen and then infested with larval Ixodes scapularis. Tick infections were determined by quantitative polymerase chain reaction of gut and salivary gland tissues. In both experimental approaches, tick infections were significantly reduced. These results suggest that subolesin appears to be a candidate vaccine antigen that may contribute to control of multiple tick species and the reduction of tick-borne pathogens.

Anaplasma marginale↗
PubMed2004

Molecular conservation of MSP4 and MSP5 in Anaplasma marginale and A. centrale vaccine strain.

Anaplasma centrale msp4 and msp5 genes were cloned and sequenced, and the recombinant proteins were expressed. The identity between Anaplasma marginale and A. centrale MSP4 was 83% in the nucleotide sequences and 91.7% in the encoded protein sequences. A. centrale msp5 nucleotide sequences shared 86.8% identity with A. marginale msp5, and there was 92.9% homology between A. centrale and A. marginale encoded amino acids of the MSP5 protein. Southern blots hybridized with probes derived from the msp4 and msp5 central regions indicate that msp4 and msp5 of A. centrale are encoded by single copy genes. Recombinant MSP4 and MSP5 fusion proteins reacted with anti-A. marginale monoclonal antibodies ANAR76A1 and ANAF16C, respectively, demonstrating the conservation of conformation-sensitive B-cell epitopes between A. centrale and A. marginale. These data demonstrate the structural and antigenic conservation of MSP4 and MSP5 in A. centrale and A. marginale. This conservation is consistent with the cross-protective immunity between A. marginale and A. centrale and supports the development of improved vaccines based upon common outer membrane proteins.

Amino Acid Sequence↗
PubMed2004

Infection of endothelial cells with Anaplasma marginale and A. phagocytophilum.

Anaplasma marginale and A. phagocytophilum are obligate intracellular, tick-borne pathogens that target erythrocytes and neutrophil granulocytes, respectively. Because ticks do not directly tap blood vessels, an intermediate tissue may mediate infection of blood cells. We considered that vascular endothelium interacts with circulating blood cells in vivo, and could be involved in pathogenesis and dissemination of the organisms. We used light and electron microscopy and immune labeling to show that A. phagocytophilum invaded rhesus (RF/6A), human (HMEC-1, MVEC), as well as bovine (BCE C/D-1b) endothelial cell lines, whereas A. marginale infected rhesus and bovine endothelial cells. A. marginale formed large intracellular inclusions that appeared smooth and solid at first, and subsequently coalesced into discrete granules. A. phagocytophilum formed numerous smaller inclusions in each cell. Within 1-3 weeks, the monolayers were destroyed, and lysed cultures were diluted onto fresh monolayers. Electron microscopy demonstrated uneven distribution of A. marginale inside large inclusions, with reticulated forms grouped more tightly than denser cells, whereas in A. phagocytophilum individual organisms appeared more evenly spaced. Specific polyclonal and monoclonal antibodies both labeled A. marginale and A. phagocytophilum in endothelial cells, and oligonucleotide primers complimentary to either A. marginale or A. phagocytophilum amplified their expected target from these cultures. In conclusion, we demonstrate that relevant microvascular endothelium is susceptible to anaplasmas in vitro and may present a link that could explain development of the immune response and persistent infection.

Anaplasma marginale↗