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Heparinized whole blood, heavily parasitized with Anaplasma marginale, was collected from 3 splenectomized oxen. Buffered lactose peptone (BLP) was added to equal volumes as a stabilizer and the mixture lyophilized in 2 ml aliquots after rapid freezing. The dried material was reconstituted with 2 ml sterile water and inoculated without delay. The product remained infective for at least 6 months when stored in an ordinary household deep-freeze unit.
Clinical and hematological changes of six Anaplasma marginale (isolated Zulia) inoculated calves (experimental group) and four healthy calves (control group) were studied during twenty and eighty days before and after infection, respectively. The behavior of the four calves used as control group was stable and no significant changes in the parameters analyzed was observed. The experimental group developed the three typical phases of illness. During the prepatent phase, which lasted a mean of 21.2 +/- 2.56 days, the animals were asymptomatic and no significant changes in the hematological values occurred, but a remarkable transitory decrease in number of lymphocytes from 6.5 x 10(6) to 3.3 x 10(6) cells/ml. The infection during the acute phase produced a highly severe effect in two animals, a severe effect in three animals and a mild effect in one. The effects observed were the following: 1) a fast decrease in haematocrite, ranging from 6 to 10%; 2) values of parasitaemia varied from 15 to 48%; 3) a greater body temperature than the control animals (40.5 vs. 38.5 degrees C); 4) a elevated heart frequency, from 60 to 110 beats/min; 5) an increase in the concentration of neotrophiles from 10 x 10(6) to 13 x 10(6) cells/ml; 6) The number of monocytes also augmented from 3 x 10(6) to 6 x 10(6) cells/ml; and 7) an important decrease of weight gain. The natural course of infection was interrupted with oxytetracycline when the haematocrite of the animal lowered to values less or equal to 10%. Then, the animals showed a rapid recovery with an undetectable parasitaemia and concomitant return to basal line of the rest of the parameters.
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.
Bacterial pathogens in the genus Anaplasma generate surface coat variants by gene conversion of chromosomal pseudogenes into single-expression sites. These pseudogenes encode unique surface-exposed hypervariable regions flanked by conserved domains, which are identical to the expression site flanking domains. In addition, Anaplasma marginale generates variants by recombination of oligonucleotide segments derived from the pseudogenes into the existing expression site copy, resulting in a combinatorial increase in variant diversity. Using the A. marginale genome sequence to track the origin of sequences recombined into the msp2 expression site, we demonstrated that the complexity of the expressed msp2 increases during infection, reflecting a shift from recombination of the complete hypervariable region of a given pseudogene to complex mosaics with segments derived from hypervariable regions of different pseudogenes. Examination of the complete set of 1183 variants with segmental changes revealed that 99% could be explained by one of the recombination sites occurring in the conserved flanking domains and the other within the hypervariable region. Consequently, we propose an 'anchoring' model for segmental gene conversion whereby the conserved flanking sequences tightly align and anchor the expression site sequence to the pseudogene. Associated with the recombination sites were deletions, insertions and substitutions; however, these are a relatively minor contribution to variant generation as these occurred in less than 2% of the variants. Importantly, the anchoring model, which can account for more variants than a strict segmental sequence identity mechanism, is consistent with the number of msp2 variants predicted and empirically identified during persistent infection.
High titered antibody from rabbits immunized with Anaplasma centrale or from cattle recovered from A. centrale infection bound predominantly to several 33-36 kDa polypeptides present in both A. centrale and the Israel-NT isolate of Anaplasma marginale. High titered bovine antibody against the Israel-NT isolate of A. marginale also reacted predominantly with A. centrale polypeptides in this size range. The immunodominance of the 33-36 kDa polypeptides and their cross-reactivity indicate that these shared epitopes may be primarily responsible for the cross-protective immunity between A. centrale and A. marginale.
Anaplasma marginale initial bodies of the Florida strain were purified from infected erythrocytes using a combination of ultrasonic disruption, nonionic detergent and differential centrifugation. Immunochemical analysis revealed at least 12 A. marginale proteins in the molecular mass (m) range 81-15 kDa with a prominent band at 38 kDa. Several of these proteins remained insoluble in the presence of nonionic detergent. Preparations of purified Anaplasma initial bodies contained negligible erythrocytic contamination, as confirmed by the minimal induction of isoantibodies against bovine blood group antigens and the absence of delayed-type hypersensitivity to erythrocytic antigens in immunized animals. A total of 33 crossbred and purebred Holstein cattle were vaccinated with either 1.5, 1.0, or 0.1 mg protein of intact initial bodies, or with 1.0 mg of solubilized Anaplasma protein. The immunogens were supplemented with 3.0 mg Quil-A saponin adjuvant and administered in 2 subcutaneous injections given at a 4-week interval. A similar number of nonvaccinated cattle served as controls. Three months after vaccination, all cattle were challenged by inoculation of 10(9) virulent A. marginale of either the homologous (Florida) or heterologous (Venezuelan) strains. Vaccinated cattle showed solid protection after homologous and heterologous challenge, characterized by parasite clearance and minimal hematocrit reductions. Initial data from four field vaccine trials revealed a reduced incidence of clinical anaplasmosis among immunized animals. Use of immunogens consisting of purified A. marginale initial bodies offers a potential immunoprophylactic approach to control of bovine anaplasmosis.
Protective immunity against the ehrlichial pathogen Anaplasma marginale has been hypothesized to require induction of immunoglobulin G2 (IgG2) antibody against outer membrane protein epitopes and coordinated activation of macrophages for phagocytosis and killing. In the present study, cell-mediated immune responses, including induction of IgG isotype switching, were characterized in calves immunized with purified outer membranes of the Florida strain of A. marginale. Importantly, these calves were subsequently shown to be protected upon experimental challenge with the Florida strain, and calves which developed the highest IgG2 titers were completely protected against infection. Peripheral blood mononuclear cells (PBMC) obtained after immunization proliferated strongly in response to both whole A. marginale homogenates and purified outer membranes, and this responsiveness persisted until the time of challenge. Responding cells were shown to be CD4(+) T cells, and CD4(+) T-cell lines cultured for 2 to 4 weeks also proliferated specifically in response to A. marginale and produced high titers of gamma interferon. The helper T-cell response included recognition of conserved epitopes, as PBMC proliferation was stimulated by the homologous Florida strain, four genetically distinct A. marginale strains, and Anaplasma ovis. The outer membrane proteins stimulating the PBMC responses in protected calves included major surface proteins (MSPs) MSP-1, MSP-2, and MSP-3, which were previously shown to induce partial protection against infection. These studies demonstrate, for the first time, potent helper T-cell responses in cattle protectively immunized with outer membranes against A. marginale challenge and identify three MSPs that are recognized by immune T cells. These experiments provide the basis for subsequent identification of the helper T-cell epitopes on MSP-1, MSP-2, and MSP-3 that are needed to evoke anamnestic antibody and effector T-cell responses elicited by protein or nucleic acid immunization.
An experimental Anaplasma marginale infection was induced in a splenectomized mule deer (Odocoileus hemionus hemionus) which persisted subclinically at least 376 days as detected by subinoculation into susceptible cattle. Anaplasma ovis was experimentally transmitted from sheep to a splenectomized and a spleen-intact mule deer, and back to sheep. The pathogenesis in deer was very similar to that seen in sheep using ovine blood inoculations.
The tick-borne rickettsia, Anaplasma marginale, causes the economically important cattle disease anaplasmosis. Once infected, cattle remain lifelong carriers. Herein, we used flow cytometry to test the efficacy of three antimicrobials; oxytetracycline, imidocarb and enrofloxacin against Virginia (VGN) or Oklahoma (OK) A. marginale isolates in short-term erythrocyte cultures. Parasite viability was assessed using the vital dye hydroethidine (HE), which is detectable when living organisms convert HE to ethidium bromide. Viability of A. marginale in selected cultures was determined by subinoculation into susceptible calves. Data were analyzed by MANOVA, Tukey-Kramer honest significant difference and Wilcoxon rank sum tests. Receiver operating characteristic (ROC) analysis was used to correlate results with culture infectivity. Enrofloxacin inhibited A. marginale in a dose dependent manner. Surprisingly, higher concentrations of imidocarb were less effective than lower concentrations against A. marginale with significant differences (P < 0.05) observed between the two isolates. Oxytetracycline was the least active drug tested. Cultures infected with the OK isolate exposed to 4.0 microg/mL enrofloxacin and those of the VGN and OK isolates exposed to 1.0 microg/mL imidocarb were sterilized. This is the first in vitro study demonstrating the efficacy of enrofloxacin against A. marginale. Furthermore, these data indicate that flow cytometry is a useful assay for screening antimicrobials against A. marginale.
Three isolates of Anaplasma marginale--Virginia (VAM), Illinois (IAM), and Florida (FAM)--were compared for infectivity for Dermacentor andersoni. The isolates were selected, in part, because of a tail-like appendage that has been demonstrated in the VAM and IAM, but not in the FAM. Ticks were exposed to the isolates as nymphs either naturally by feeding on a calf with anaplasmosis or artificially by percutaneous inoculation with infected bovine erythrocytes. They were examined for infectivity after molting to the adult stage by determining their capability to transmit the disease to susceptible calves and by demonstrating colonies in tick gut sections. Only those ticks exposed to the VAM proved to be infected with A marginale; ticks naturally exposed and those artificially infected with this isolate transmitted the disease to susceptible calves. Colonies of A marginale were observed only in gut tissues of ticks naturally infected with VAM. The IAM (appendage present) and FAM (appendage absent) could not be found in ticks exposed by either method, indicating that factors other than the presence of inclusion appendages may be involved in infection of ticks by A marginale.
Partially purified Anaplasma marginale initial bodies were cultivated in a cell-free system in the presence of [3-14C]pyruvate for 24 or 48 h. Experiments showed that a significant portion of the pyruvate supplied to the cultures was incorporated into initial body components. Label incorporation was reduced by 72% in the presence of oxytetracycline. Fractionation and chromatography of the organisms revealed radioactive incorporation as alanine. This is the first report of de novo amino acid synthesis by A. marginale demonstrating that the rickettsia is capable of using pyruvate, an erythrocyte glycolytic product, in its metabolism.
Four Anaplasma marginale-infected splenectomized calves with greater than 25% parasitized erythrocytes received enrofloxacin at 12.5 mg/kg SC twice, 48 hours apart. Two infected splenectomized calves were designated as untreated controls. A precipitous decline in percent parasitized erythrocytes from 39.13% to less than 1% was observed over 12 days following treatment. However, a self-limiting recrudescence of A. marginale parasites was observed within 30 days after treatment. Untreated control calves became moribund and were euthanized. These data indicate that the regimen of enrofloxacin tested herein ameliorates, but does not eliminate, A. marginale infections in splenectomized calves.
Immunization of cattle with a purified Anaplasma marginale major surface protein, AmF36, induced protection against homologous challenge with the Florida isolate. Similarly, immunized cattle were protected from challenge with the antigenically and structurally distinct Washington-O isolate of A. marginale. The degree of protection in AmF36-immunized cattle varied from complete prevention of rickettsemia to significant delay in the onset of rickettsemia compared with control immunized cattle. A single AmF36 vaccinate was not protected against homologous challenge despite development of a strong antibody response. Immunoprecipitation of A. marginale proteins with a monoclonal antibody to AmF36 identified minor molecular size heterogeneity in this protein from different isolates, including the Florida and Washington-O isolates. The apparent molecular size of this surface protein in the Florida isolate was 36 kilodaltons, whereas the analogous proteins in Washington-O and four other isolates of A. marginale from the United States had molecular masses of 33 to 34 kilodaltons. Significantly, the surface-exposed peptides of these proteins appear to be conserved among the different isolates. These results demonstrate the potential of AmF36 as a subunit immunogen for bovine anaplasmosis and indicate a structural basis for its cross-protective ability.
Colonies of Anaplasma marginale Theiler were studied in midgut epithelial cells of adult Dermacentor andersoni Stiles that were infected as nymphs. The colonies were demonstrated in plastic sections (1 micrometer) and frozen sections (6 micrometer) with Mallory's stain. Colonies were also demonstrated in frozen sections, using direct fluorescent antibody technique. Colonies were not observed in plastic or frozen sections stained by the Giménez method. In all preparations, the colonies were usually found near the basement membrane of the midgut tissue.
An inoculum of Anaplasma marginale was given (IV) to a splenectomized domestic goat and then was serially passed through 4 additional goats. The results indicated that goats can be latent carriers at 75 days after inoculation, but not at 208 days. There was no evidence of serial passage of A marginale in goats.
Anaplasma marginale is an obligate intraerythrocytic rickettsial pathogen (order, Rickettsiales: family, Anaplasmataceae) that causes bovine anaplasmosis. This disease is widely distributed in tropical and sub-tropical regions of the world and causes important economic losses to cattle production. Major surface protein (MSP)1a (msp1alpha gene) is one of the six MSPs identified on A. marginale from cattle, whose sequence and size vary according to the number of tandem 28- to 29-amino acid repeats. This study characterized the msp1alpha and msp4 genes obtained from three distinct Brazilian herds from the State of Paraná. Three strains of the msp1alpha and one strain of the msp4 gene were sequenced. The strains evaluated revealed PCR products of different size, representing three, five and six internal repeats. Sequence analyses confirmed the number of tandem sequence copies and revealed a high degree of sequence identity with strains from other Brazilian States, as well as strains from the USA, Europe and Israel. The msp1alpha DNA and amino acid sequences from A. marginale and DNA sequences of msp4 strains did not reveal distinct phylogeographical segregation. However, the amino acid sequences of msp4 demonstrated definite phylogeographical relationship. These results suggest that the amino acid sequences of msp4 should be used for phylogenetic identification of A. marginale strains and may be an important tool for the epidemiology and control of anaplasmosis. Additionally, the close similarity of the Paraná strains of A. marginale with strains from USA, Europe and Asia may reflect the introduction of these genes during the development of the Brazilian bovine herd.
The polymerase chain reaction (PCR) was used to detect Anaplasma marginale in hemolymph collected from live Dermacentor andersoni Stiles ticks. Hemolymph was collected from severed legs of male and female ticks exposed to A. marginale as either nymphs or adults. Heat treatment was found to be the optimum method of hemolymph preparation for PCR. Hemolymph samples were collected and pooled from adult ticks exposed as nymphs on days 0-10 of feeding on a susceptible calf. For male and female ticks exposed as adults, samples were collected as ticks fed 7 d on an infected calf, while being held 9 d between feedings, and during a second feeding of 10 d (or to repletion) when they transmitted the parasite. Hemolymph samples were collected from uninfected ticks at the same times to serve as controls. Anaplasma marginale DNA was amplified with primers BAP-2 (5'-GTATGGCACGTAGTCTTGGGATCA-3') and AL34S (5'-CAGCAGCAGCAAGACCTTCA-3'), which flank a 409-bp fragment of the A. marginale Florida isolate msp1 beta gene. Infected tick hemolymph was PCR-positive for A. marginale at all collection times, including unfed adults infected as nymphs and previously unexposed adults that fed on infected calves for only 1 d. The PCR-based assay of tick hemolymph proved to be a sensitive method for identification of infected ticks, potentially without killing them; it would be well suited for identification of laboratory- or field-infected ticks that could then be used for further studies. The primers used in this assay were also found specific when tested with species of 18 different genera, and universal for 7 A. marginale isolates from diverse geographical areas of the United States.
Anaplasma marginale-infected erythrocytes were examined for the presence of maturation, isolate-restricted, and isolate-common antigens by indirect immunofluorescence with monoclonal antibodies. A panel of 18 monoclonal antibodies was used; none of the antibodies reacted with Anaplasma ovis, Babesia bigemina, babesia bovis, Trypanosoma brucei, Trypanosoma congolense, or uninfected bovine erythrocytes. Antigens common to all six A. marginale isolates were detected by nine antibodies. Single isolates from Florida, Southern Idaho, Northern Texas, and Virginia and two isolates from Washington state had four patterns of reactivity with a second panel of nine antibodies. Antigenically distinct stages were not detected, as sequential smears taken daily during acute infection had the same pattern of reactivity. The results demonstrate antigenic heterogeneity among isolates of A. marginale and the presence of common antigens. This information allows grouping of different isolates and, more importantly, provides a method for the identification and isolation of common antigens for diagnostic tests.