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Human granulocytic anaplasmosis (HGA), an emerging disease of public health concern in many areas of the world, is caused by Anaplasma phagocytophilum.Small animal models of A phagocytophilumin laboratory mice have been developed and used to study the pathogenesis of HGA. In this study, we characterized the pathologic changes in acute infection of C3H/HeJ mice experimentally infected with the NY18 isolate of A phagocytophilum.Although no clinical signs were noted, acute infection was associated with gross splenomegaly, microscopic inflammatory lesions in the lung and liver, hyperplastic lesions on the spleen, and clinical pathology abnormalities including neutropenia and monocytosis. This study emphasizes the use of well-defined animal models as a valuable tool for the study of A phagocytophiluminfections.

ABSTRACTIn the context of a serosurvey conducted on the Anaplasma marginaleprevalence in Swiss cattle, we suspected that a serological cross-reactivity between A. marginaleand A. phagocytophilummight exist. In the present study we demonstrate that cattle, sheep and horses experimentally infected with A. phagocytophilumnot only develop antibodies to A. phagocytophilum(detected by immunofluorescent-antibody assay) but also to A. marginale(detected by a competitive enzyme-linked immunosorbent assay). Conversely, calves experimentally infected with A. marginalealso developed antibodies to A. phagocytophilumusing the same serological tests. The identity of 63% determined in silico within a 209-amino-acid sequence of major surface protein 5 of an isolate of A. marginaleand one of A. phagocytophilumsupported the observed immunological cross-reactivity. These observations have important consequences for the serotesting of both, A. marginaleand A. phagocytophiluminfection of several animal species. In view of these new findings, tests that have been considered specific for either infection must be interpreted carefully.

The tick-borne intracellular pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) develops persistent infections in cattle and tick hosts. While erythrocytes appear to be the only site of infection in cattle, A. marginale undergoes a complex developmental cycle in ticks and transmission occurs via salivary glands during feeding. Many geographic isolates occur that vary in genotype, antigenic composition, morphology and infectivity for ticks. In this chapter we review recent research on the host–vector–pathogen interactions of A. marginale. Major surface proteins (MSPs) play a crucial role in the interaction of A. marginale with host cells. The MSP1a protein, which is an adhesin for bovine erythrocytes and tick cells, is differentially regulated and affects infection and transmission of A. marginale by Dermacentor spp. ticks. MSP2 undergoes antigenic variation and selection in cattle and ticks, and contributes to the maintenance of persistent infections. Phylogenetic studies of A. marginale geographic isolates using msp4 and msp1α provide information about the biogeography and evolution of A. marginale: msp1α genotypes evolve under positive selection pressure. Isolates of A. marginale are maintained by independent transmission events and a mechanism of infection exclusion in cattle and ticks allows for only the infection of one isolate per animal. Prospects for development of control strategies by use of pathogen and tick-derived antigens are discussed. The A. marginale/vector/host studies described herein could serve as a model for research on other tick-borne rickettsiae.

ABSTRACTThe rickettsial pathogen Anaplasma marginaleexpresses a variable immunodominant outer membrane protein, major surface protein 2 (MSP2), involved in antigenic variation and long-term persistence of the organism in carrier animals. MSP2 contains a central hypervariable region of about 100 amino acids that encodes immunogenic B-cell epitopes that induce variant-specific antibodies during infection. Previously, we have shown that MSP2 is encoded on a polycistronic mRNA transcript in erythrocyte stages of A. marginaleand defined the structure of the genomic expression site for this transcript. In this study, we show that the same expression site is utilized in stages of A. marginaleinfecting tick salivary glands. We also analyzed the variability of this genomic expression site in Oklahoma strain A. marginaletransmitted from in vitro cultures to cattle and between cattle and ticks. The structure of the expression site and flanking regions was conserved except for sequence that encoded the MSP2 hypervariable region. At least three different MSP2 variants were encoded in each A. marginalepopulation. The major sequence variants did not change on passage ofA. marginalebetween culture, acute erythrocyte stage infections, and tick salivary glands but did change during persistent infections of cattle. The variant types found in tick salivary glands most closely resembled those present in bovine blood at the time of acquisition of infection, whether infection was acquired from an acute or from a persistent rickettsemia. These variations in structure of an expression site for a major, immunoprotective outer membrane protein have important implications for vaccine development and for obtaining an improved understanding of the mechanisms of persistence of ehrlichial infections in humans, domestic animals, and reservoir hosts.

Anaplasma marginale, a tick-borne rickettsial pathogen of cattle, infects bovine erythrocytes, resulting in mild to severe hemolytic disease that causes economic losses in domestic livestock worldwide. Recently, the Virginia isolate of A. marginale was propagated in a continuous tick cell line, IDE8, derived from embryonic Ixodes scapularis. Development of A. marginale in cell culture was morphologically similar to that described previously in ticks. In order to evaluate the potential of the cell culture-derived organisms for use in future research or as an antigen for serologic tests and vaccines, the extent of structural conservation of the major surface proteins (MSPs) between the cell culture-derived A. marginale and the bovine erythrocytic stage, currently the source of A. marginale antigen, was determined. Structural conservation on the tick salivary-gland stage was also examined. Monoclonal and monospecific antisera against MSPs 1 through 5, initially characterized against erythrocyte stages, also reacted with A. marginale from cell culture and tick salivary glands. MSP1a among geographic A. marginale isolates is variable in size because of different numbers of a tandemly repeated 28- or 29-amino-acid peptide. The cell culture-derived A. marginale maintained the same-size MSP1a as that found on the Virginia isolate of A. marginale in bovine erythrocytes and tick salivary glands. Although differences were observed in the polymorphic MSP2 antigen between culture and salivary-gland stages, MSP2 did not appear to vary, by two-dimensional gel electrophoresis, during continuous passage in culture. These data show that MSPs of erythrocyte-stage A. marginale are present on culture stages and may be structurally conserved during continuous culture. The presence of all current candidate diagnostic and vaccine antigens suggests that in vitro cultures are a valuable source of rickettsiae for basic research and for the development of improved diagnostic reagents and vaccines against anaplasmosis.

Fingerling channel catfish Ictalurus punctatusexposed to simulated ultraviolet-B radiation at an average daily dose of 2.9 J/cm2were quite sensitive to the radiation. After a 24-h exposure, thinning of the most dorsal epidermis frequently was accompanied by edema. Compared with epidermis of unexposed fish, mucous cells in exposed fish were less superficial and club cells were less numerous both dorsally and high on the lateral surface of the body. Sunburn cells with pyknotic nuclei were evident in the epidermis of exposed fish. Among fish exposed for 48 h, focal necrosis and sloughing of the outer epidermal layer were widespread. A methanol-extractable skin substance that is associated with resistance to sunburn in other fish species was not detected in channel catfish.

ABSTRACTAnaplasma marginale, a tick-borne rickettsial pathogen of cattle, infects bovine erythrocytes, resulting in mild to severe hemolytic disease that causes economic losses in domestic livestock worldwide. Recently, the Virginia isolate of A. marginalewas propagated in a continuous tick cell line, IDE8, derived from embryonic Ixodes scapularis. Development of A. marginalein cell culture was morphologically similar to that described previously in ticks. In order to evaluate the potential of the cell culture-derived organisms for use in future research or as an antigen for serologic tests and vaccines, the extent of structural conservation of the major surface proteins (MSPs) between the cell culture-derived A. marginaleand the bovine erythrocytic stage, currently the source of A. marginaleantigen, was determined. Structural conservation on the tick salivary-gland stage was also examined. Monoclonal and monospecific antisera against MSPs 1 through 5, initially characterized against erythrocyte stages, also reacted with A. marginalefrom cell culture and tick salivary glands. MSP1a among geographic A. marginaleisolates is variable in size because of different numbers of a tandemly repeated 28- or 29-amino-acid peptide. The cell culture-derivedA. marginalemaintained the same-size MSP1a as that found on the Virginia isolate of A. marginalein bovine erythrocytes and tick salivary glands. Although differences were observed in the polymorphic MSP2 antigen between culture and salivary-gland stages, MSP2 did not appear to vary, by two-dimensional gel electrophoresis, during continuous passage in culture. These data show that MSPs of erythrocyte-stage A. marginaleare present on culture stages and may be structurally conserved during continuous culture. The presence of all current candidate diagnostic and vaccine antigens suggests that in vitro cultures are a valuable source of rickettsiae for basic research and for the development of improved diagnostic reagents and vaccines against anaplasmosis.

Major surface proteins of Anaplasma marginale are vaccine candidates. We recently demonstrated that immunization of calves with outer membranes of the Florida strain of A. marginale resulted in protective immunity that correlated with a memory CD4(+) T-lymphocyte response specific for major surface protein 1 (MSP-1), MSP-2, and MSP-3 (W. C. Brown, V. Shkap, D. Zhu, T. C. McGuire, W. Tuo, T. F. McElwain, and G. H. Palmer, Infect. Immun. 66:5406-5413, 1998). As immunogens, these proteins have been shown to induce complete or partial protection against homologous challenge. To further define the T helper (Th) cell response to these and other A. marginale antigens and to determine conservation of Th cell epitopes among genetically distinct A. marginale strains, Th cell clones obtained prior to challenge from three immunized calves were characterized for antigen-specific responses. Nine distinct antigenic profiles were defined by 11 Th cell clones derived by stimulation with the Florida strain. Several clones responded to MSP-2, MSP-3, or both. All of these MSP-2- or MSP-3-specific clones and the majority of other clones that did not respond to MSPs recognized all bovine blood-passaged strains of A. marginale. These results demonstrate conservation of certain Th cell epitopes between MSP-2 and MSP-3 and show that Th cell epitopes in MSP-2, MSP-3, and undefined antigens are conserved among strains of A. marginale. Of seven clones that responded to the blood-passaged Virginia strain, two did not recognize antigen prepared from this strain cultured in tick cells, suggesting differences in the antigenic composition between these stages. Analysis of the cytokines expressed by the Th cells revealed that all clones expressed gamma interferon and tumor necrosis factor alpha, and most coexpressed interleukin-4. Our results provide a rationale for identifying Th cell epitopes conserved among different strains of A. marginale for inclusion in a nucleic acid or recombinant protein vaccine.

Lahontan cutthroat trout Oncorhynchus clarki henshatviwere sensitive to simulated solar ultraviolet-B radiation (UVB) and exhibited grossly visible signs of sunburn upon exposure. Razorback suckers Xyrauchen texanus, however, were tolerant to simulated solar UVB and showed no grossly visible signs of exposure. Cutthroat trout also had considerably less of an unidentified, possibly photoprotective, substance in the skin than did razorback suckers. In an attempt to characterize the cellular response to simulated solar UVB exposure in the skin of these two species, we examined sections from UVB-exposed fish by light and electron microscopy. Cutthroat trout showed grossly visible signs of sunburn by 48 h. Histologic observations included a sloughing of the mucous cells, necrosis and edema in the epidermis and dermis, and, in some cases, secondary fungal infections. Razorback suckers did not show any visible signs of sunburn during 72 h of experimental exposure. Histologic analyses revealed that cell necrosis had occurred, but the severe necrosis and sloughing noted in cutthroat trout was not observed. An increase in epidermal thickness, apparently due to hypertrophy and hyperplasia of large PAS-negative cells, occurred in the razorback suckers. These cells contained a large central region of low electron density and appeared to be club cells. In some, extensive interdigitation of the electron-lucent cytoplasm with adjacent epithelial cell margins occurred. Near the surface of the epidermis these cells were larger and the interface with epithelial cells lacked complex interdigitation. These cells may contain the substance that appears to protect razorback suckers against UV-B radiation.

Serologic diagnosis of anaplasmosis is currently done by the complement-fixation, ELISA, and card agglutination tests. These tests have utilized A. marginaleharvested from bovine erythrocytes as antigen which is often contaminated with erythrocyte stroma. We are currently testing A. marginalepropagated in a Ixodes scapulariscell line as antigen for serologic tests. In this study, we report the use of the cell culture-derived A. marginaleas antigen for development of a rapid, semi-automated latex agglutination test. Diluted serum and latex (polystyrene microspheres), sensitized with cell culture-derived A. marginaleproteins, were dispensed into 96-well microtiter plates. An initial reading of light transmission was recorded by a computer-interfaced scanning autoreader. After 30 minutes, the plates were mixed and read a second time, recording the delta light transmittance. The sensitized latex microspheres (latex) agglutinated in the presence of A. marginale antibodies, thus producing an increase in light transmittance. In preliminary tests, 724977 of the sera were positive for A. marginaleantibodies with an apparent agreement of 83.3 when compared with the complement-fixation test. Sensitization and sera dilution buffers were shown to have a marked effect on the sensitivity and specificity of this assay. Results will be presented on the optimization of buffers and the testing of sera from experimentally and field-infected cattle.

An obligate intracellular rickettsial organism isolated from an aborted bovine fetus was studied in bovine turbinate and mouse macrophage cell cultures with light and electron microscopy. Development of the organism was similar in both cell types. The organism replicated within cytoplasmic vacuoles in a developmental cycle that resembled that of both the ehrlichiae and chlamydiae. The inoculum contained only electron-dense forms, which infected cells within 2 h postinoculation by adhering to cell membranes at thickened areas that appeared to be coated pits and then being endocytosed. A striking feature occurred next as the organisms became surrounded by host cell mitochondria and, by light microscopy, appeared to have halos. During this intimate association with mitochondria, the electron-dense organisms changed into large reticulated forms that began to divide by binary fission. These large forms were often in direct contact with mitochondrial membranes. The organisms continued to divide by binary fission, and host cells contained large cytoplasmic inclusions of reticulated organisms. The reticulated organisms gradually changed into electron-dense forms that were released from degenerated host cells.

A microsporidian of the genus Loma was noted in the gills of rainbow trout Oncorhynchus mykissfrom a state hatchery (Buford Trout Hatchery) in Georgia. Mortalities of varying severity occur at this hatchery every fall, and the microsporidian was noted during an experiment from August 1991 to January 1992 to determine the effects of water source on disease. Infections first appeared to be systemic in the October sample; xenomas were observed in heart, spleen, and peripheral vessel walls. The presence of unidentified intracellular material preceded the appearance of xenomas in all tissues, but whether this material was associated with inflammation or represented immature stages of the parasite has yet to be determined. These structures were also noted in the intestine and liver, although xenomas were not noted in these organs. Mature xenomas did not elicit an inflammatory response but appeared to be short-lived. When the xenoma wall ruptured and released spores, an inflammatory response was again observed. The prevalence and severity of the infection were determined in fish maintained in troughs with well water, Chattahoochee River water, or hatchery (treated river) water. The infection tended to be more prevalent and more severe in fish maintained in the hatchery or river water than in those maintained in the well water. Stress induced by poor water quality may increase mortality from this parasite. This report extends the range of Lomasp. into the eastern United States.

Epitopes of major surface proteins of the intraerythrocytic cattle stage of Anaplasma marginale were demonstrated in the midgut stage of the organism within the infective tick host Dermacentor andersoni. These proteins were common to all A. marginale isolates tested and at all stages of parasitemia. Sera from cattle immunized with the tick midgut stage of A. marginale immunoprecipitated multiple-erythrocyte-stage proteins, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major proteins recognized (primarily greater than 14 and less than 200 kilodaltons [kDa]) included two major-erythrocyte-stage surface proteins of 36 and 105 kDa molecular size. To confirm the presence of common tick and erythrocyte A. marginale antigens with the immunized cattle sera, we purified the 36-kDa erythrocyte-stage protein by monoclonal immunoaffinity chromatography and developed an enzyme-linked immunosorbent assay based on the purified protein. All sera from cattle immunized with tick-stage A. marginale and cattle infected with various isolates of A. marginale developed antibodies to the 36-kDa protein. The potential immunoprophylactic, diagnostic, and epidemiologic value of the major epitopes common to both the invertebrate and mammalian stages of A. marginale, especially the 36-kDa protein, is discussed.

Bovine pineal glands were extracted with acetic acid and partially purified on Sephadex G-25. Three fractions, F3, F4 and F5, were each administered to cycling female rats for 4, 2 or 1 day prior to ovulation to determine their effects on fertility, ovulation and the pro-oestrous surge of luteinizing hormone (LH). The incidence of pregnancy and the mean number of foetuses were reduced in animals after treatment with F3 or F4 but not in those treated with F5. Each of the F3 and F4 fractions significantly reduced the mean number of ova shed and the pre-ovulatory concentration of serum LH. The F5 fraction caused a moderate reduction of the pro-oestrous rise of serum LH but had no significant effect on ovulation. The data suggest that fertility in rats can be reduced by more than one substance of low molecular weight present in bovine pineal extracts through a modifying effect on LH secretion and subsequent ovulation.

Anaplasma marginale, a tick-borne rickettsia that infects erythrocytes of cattle, occurs within a parasitophorous vacuole or inclusion body. A tail-like inclusion appendage, composed of multiple filaments, occurs in association with the inclusion body membrane. The composition and function of the inclusion appendage have not been determined. In this study, theA. marginaleinclusion appendage in bovine erythrocytes was found to be composed of actin filaments as determined by labeling with rhodamine-conjugated phalloidin. Electron microscopy studies revealed that theA. marginaleinclusion appendages differed from F-actin tails reported previously in association with other pathogens in eukaryotic cells because these highly ordered structures were organized into regularly occurring striations, and the appendages were adhered directly to the parasitophorous vacuole membrane. In addition, actin appendages have not been described previously in erythrocytes. The potential role of the inclusion appendage associated withA. marginalein bovine erythrocytes and recently fed ticks is discussed.

An ear tick, Otobius megnini (Dugés) recovered from a child who had serologic evidence of ehrlichiosis, was examined for Ehrlichia species microscopically and by inoculation into a susceptible dog; no evidence of infection was found in the tick. Experimental transmission of E. canis by laboratory-reared O. megnini was attempted; neither transstadial nor transovarial transmission occurred.