Major surface protein 1a effects tick infection and transmission of Anaplasma marginale.

Anaplasma marginale, an ehrlichial pathogen of cattle and wild ruminants, is transmitted biologically by ticks. A developmental cycle of A. marginale occurs in a tick that begins in gut cells followed by infection of salivary glands, which are the site of transmission to cattle. Geographic isolates of A. marginale vary in their ability to be transmitted by ticks. In these experiments we studied transmission of two recent field isolates of A. marginale, an Oklahoma isolate from Wetumka, OK, and a Florida isolate from Okeechobee, FL, by two populations of Dermacentor variabilis males obtained from the same regions. The Florida and Oklahoma tick populations transmitted the Oklahoma isolate, while both tick populations failed to transmit the Florida isolate. Gut and salivary gland infections of A. marginale, as determined by quantitative PCR and microscopy, were detected in ticks exposed to the Oklahoma isolate, while these tissues were not infected in ticks exposed to the Florida isolate. An adhesion-recovery assay was used to study adhesion of the A. marginale major surface protein (MSP) 1a to gut cells from both tick populations and cultured tick cells. We demonstrated that recombinant Escherichia coli expressing Oklahoma MSP1a adhered to cultured and native D. variabilis gut cells, while recombinant E. coli expressing the Florida MSP1a were not adherent to either tick cell population. The MSP1a of the Florida isolate of A. marginale, therefore, was unable to mediate attachment to tick gut cells, thus inhibiting salivary gland infection and transmission to cattle. This is the first report of MSP1a being responsible for effecting infection and transmission of A. marginale by Dermacentor spp. ticks. The mechanism of tick infection and transmission of A. marginale is important in formulating control strategies and development of improved vaccines for anaplasmosis.

Development of Anaplasma ovis (Rickettsiales: Anaplasmataceae) in male Dermacentor andersoni (Acari: Ixodidae) transferred from infected to susceptible sheep.

The development of Anaplasma ovis was studied in Dermacentor andersoni males transferred from infected to susceptible sheep. Laboratory-reared male D. andersoni were allowed to feed for 6 d on a sheep with ascending A. ovis parasitemia. The ticks were removed and held at room temperature in a humidity chamber for 6 d, after which they were allowed to feed on five susceptible sheep for 1, 3, 5, 7, or 9 d. Gut and salivary glands were collected from ticks during the 21-d experiment and examined with light and electron microscopy. Anaplasmosis developed in all susceptible sheep. Colonies of A. ovis were first observed in midgut epithelial cells on the 3rd d ticks fed on the infected sheep, and infection persisted in gut cells throughout the experiment. The first colonies contained one large organism that subsequently gave rise to many reticulated ones, which became electron dense over time. After ticks were transferred to susceptible sheep and began the second feeding, individual A. ovis organisms were found from days 3-9 in muscle cells on the hemocoel side of the gut basement membrane. However, colonies did not develop in these cells, and the host cells did not hypertrophy as did cells similarly infected with A. marginale. A final site of development of A. ovis was in salivary glands. Individual organisms were first seen in acinar cells on the first day that ticks fed on the second calves, and salivary gland infections persisted throughout the 9-d feeding period. Colonies of A. ovis developed in salivary gland acinar cells and organisms within these colonies were initially electron lucent but became electron dense. Multiple colonies often were observed within salivary gland cells and often contained organisms in various stages of development.

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.

Developmental studies of Anaplasma marginale (Rickettsiales:Anaplasmataceae) in male Dermacentor andersoni (Acari:Ixodidae) infected as adults by using nonradioactive in situ hybridization and microscopy.

The development of Anaplasma marginale Theiler was studied in ticks using a nonradioactive in situ hybridization method developed in our laboratory. Male Rocky Mountain wood ticks, Dermacentor andersoni Stiles, were infected intrastadially by allowing them to feed for 7 d on an infected calf (acquisition feeding). The ticks were then removed and held in a humidity chamber for 5 d before being fed on a 2nd susceptible call for 10 d (transmission feeding). Two groups of 10 ticks were collected daily during the 22-d experiment. In one group one-half of each tick was processed and embedded in paraffin and in the other group one-half of each tick was embedded in LR White for in situ hybridization. The companion tick halves from each group were fixed and embedded in Dow Epoxy Resin resin for routine light and electron microscopy. As detected by in situ hybridization on LR White- and paraffin-embedded sections and by microscopy, initial infection of A. marginale in ticks occurred in gut tissues either on the 7th d of acquisition feeding or the 1st d of the held period and infection persisted throughout transmission feeding. The highest number of ticks with gut infection was observed on the 5th d of transmission feeding. Salivary glands became infected with A. marginale on the 1st day of transmission feeding and remained infected throughout the transmission feeding period. Peak infection was observed on day 4 of transmission feeding. After the beginning of transmission feeding, A. marginale infection was also observed in interstitial, reproductive, skeletal muscle, fat body, and Malpighian tubule tissues. Although A. marginale infection of ticks clearly originates in midgut epithelial cells, many tissues eventually become infected during transmission feeding, resulting in a generalized infection. The infection of multiple tissues may contribute to the ability of A. marginale infection to persist in intrastadially infected male ticks.

Establishment of the tick (Acari:Ixodidae)-borne cattle pathogen Anaplasma marginale (Rickettsiales:Anaplasmataceae) in tick cell culture.

Anaplasma marginale is a tick-borne rickettsia that causes bovine anaplasmosis worldwide. Despite its importance, A. marginale has thus far not been established in a continuous culture system. We have propagated A. marginale continuously for the 1st time in a tick cell line derived from the black-legged tick, Ixodes scapularis Say, using infected bovine blood as the inoculum. Erythrocytic stages invaded the tick cells and multiplied in membrane-lined vacuoles to form colonies typical of those observed in naturally infected ticks as demonstrated by light and electron microscopy. The rickettsiae have been passaged serially for 3 yr and have been cryopreserved in liquid nitrogen. Antigens present in A. marginale from tick cell culture were recognized by bovine immune serum against the blood stages of A. marginale. A. marginale grown in this tick cell line was infective for calves, and male ticks fed on the calves transmitted A. marginale to a susceptible calf. The ability to culture A. marginale removes a major impediment to the study of Anaplasma biology in vitro, and will enhance development of vaccines and diagnostic tests.

Antigen recognition on Anaplasma marginale and bovine erythrocytes: an electron microscopy study.

The reactivity of sera from Anaplasma marginale-infected bovine with red blood cells and with purified anaplasma bodies was analyzed by electron immunomicroscopy. Red blood cells from non-infected and from anaplasma-infected cows and A. marginale bodies separated from parasitized erythrocytes, were incubated with control, pre-immune and immune sera followed by anti-bovine IgG-Peroxidase. Immune sera from cows infected with the venezuelan and Florida isolate reacted with red blood cell membranes from normal and infected bovines, while sera from non-infected cows did not. The immune sera also recognized epitopes localized on the cell wall, membrane and on unidentified intracellular structures of the purified anaplasma bodies. Thus, we propose that A. marginale infection may cause structural and biochemical modifications of the plasma membrane of the bovine red blood cells during its intraerythrocytic cycle. This in turn could elicit an autoimmune type of response against its own cells that would stimulate erythrophagocitosis. The strong reactivity of the immune sera with the Anaplasma bodies suggests that the bovine immune system also recognizes epitopes located on the parasite.

Ultrastructure of Anaplasma marginale with an inclusion appendage, isolated in Minas Gerais State, Brazil.

This is the first report on the occurrence and isolation of a strain of Anaplasma marginale with an inclusion appendage in Brazil. The inclusion appendage presented longitudinal electron-dense striations and did not originate directly from the body of the rickettsia but from an electron-dense complex located at the junction of the inclusion membrane and inclusion appendage. The inclusion appendage remained in the host cell after the Anaplasma inclusion appeared to be leaving the red blood cell. Other ultrastructures of this rickettsia are described and its epidemiological importance is discussed.

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.

Establishment and characterization of an Oklahoma isolate of Anaplasma marginale in cultured Ixodes scapularis cells.

Anaplasma marginale is a tick-borne hemoparasite of cattle worldwide. The Virginia isolate of A. marginale was propagated previously in a cell line derived from embryos of the tick, Ixodes scapularis. The cultured Anaplasma (VA-tc) was passaged continuously for over 4 years and retained its infectivity for cattle and antigenic stability. We report herein the continuous in vitro cultivation of a second isolate of A. marginale derived from a naturally infected cow in Oklahoma (OK-tc). Blood from the infected cow was subinoculated into a splenectomized calf and blood collected at peak parasitemia was frozen, thawed and used as inoculum on confluent tick cell monolayers. Colonies of Anaplasma were apparent in low numbers at 9 days post exposure (PE) and infection in monolayers reached 100% by 4-5 weeks PE. Cultures were passaged by placing supernatant onto fresh tick cell monolayers at a dilution of 1:5 or 1:10. By the third passage development of the OK-tc was similar to that of the VA-tc and a 1:5 dilution resulted in 100% infection in 10-12 days. Inoculation of OK-tc into a splenectomized calf caused clinical anaplasmosis and Dermacentor ticks that fed on this calf transmitted the organism to a second susceptible calf. Major surface proteins (MSPs) 1-5 of the OK-tc were compared with homologous proteins present on VA-tc and the erythrocytic stage of the Oklahoma isolate. The MSPs 1, 2, 4, 5 were conserved on the OK-tc but there was evidence for structural variation in MSP3 between the cultured and erythrocytic stage of Anaplasma. MSP2 and MSP3 were the major proteins recognized by serum from infected cattle. Two-dimensional gels also identified positional differences between VA-tc and OK-tc in MSP2 and MSP3. The OK-tc may have potential to be used as antigen for development of an improved vaccine for anaplasmosis in the South Central United States.

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.

Antigenic characterization of morphologically distinct Anaplasma marginale isolates using a panel of monoclonal antibodies.

The present study, describes the antigenic characterization of a Brazilian isolate of Anaplasma marginale with appendage (tail). A panel of monoclonal antibodies (McAbs) was produced and tested by the indirect fluorescent antibody test (IFAT), ELISA and Western blotting, and used to characterize two isolates of A. marginale (one with appendage and another without appendage). Among the clones produced, eight recognized antigenic proteins, with molecular weights varying from 18.4 to 66kDa. In Western blotting, the McAb reacted against a 45kDa antigen, which was shown, by the IFAT, to be located in the tail. Immunocytochemistry confirmed the tail specificity of the monoclonal reacting against the 45kDa antigen. The panel of McAb produced has a potential use in discriminating morphologically distinct A. marginale isolates. The present study, demonstrates the occurrence of antigenic diversity among Brazilian isolates of A. marginale.

Multiple infection in bovines from the tropics: observation of blood parasites by scanning and transmission electron microscope.

Blood samples from a splenectomized bovine, experimentally inoculated with blood from a field cow living in southwestern Venezuela, were processed for transmission and scanning electron microscopy. The blood sample showed multiple infection with hemoparasites of the genera Anaplasma marginale, Eperythrozoon wenyonii and Trypanosoma vivax. Scanning electron microscope showed that the blood from bovines with multiple infection had profound deformation in knob-like protruding structures with reduced cellular volume similar to echinocyte red blood cells. E. wenyonii parasites appear associated with the membrane, grouped in shallow to severe invaginations at the surface of the erythrocytes. The morphology of the parasites is predominantly rod-like; they also appear as coccoid-shaped and bifurcate or triskelion-shaped organisms. The organisms are present in pairs or clusters. T. vivax appeared with double flagella, which indicates active cellular division and infection processes. Transmission electron microscope study showed erythrocytes infected with intracytoplasmic bodies of A. marginale and with E. wenyonii embedded in the external membrane cell, with mature, juvenile and dividing forms present.

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.

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.

Morphologic characteristics of colonies of Anaplasma marginale Theiler in midgut epithelial cells of Dermacentor andersoni Stiles.

Colonies of Anaplasma marginale Theiler were studied in midgut epithelial cells of adult Dermacentor andersoni Stiles that were infected as nymphs. Colonies were categorized by light microscopy into 5 morphologic types that appear to represent stages in a developmental sequence. The mean diameter of the colony types ranged from 5.64 micrometer to type 1 colonies to 10.49 micrometers of type 5 colonies. Representatives of each colony type were selected by light microscopy and were sectioned for examination by electron microscopy. The morphologic features of individual A marginale organisms within colony types varied and included: (i) small electron-dense forms, (ii) larger reticulated forms, (iii) pleomorphic reticulated forms, and (iv) small particles. Types 1 and 2 colonies contained small, electron-dense forms. Type 3 colonies contained electron-dense forms, early reticulated forms, and small particles that were often outside of the parasite's limiting membrane. Type 4 colonies contained many reticulated forms that often had small particles within cell membranes. Type 5 colonies contained fewer well-formed reticulated forms than did type 4 and, in some cases, large masses of pleomorphic reticulated forms.

Development of Anaplasma marginale in male Dermacentor andersoni transferred from parasitemic to susceptible cattle.

The development and transmission of Anaplasma marginale was studied in Dermacentor andersoni males. Laboratory-reared male D andersoni were allowed to feed for 7 days on a calf with ascending A marginale parasitemia. The ticks were then held in a humidity chamber for 7 days before being placed on 2 susceptible calves. Anaplasmosis developed in the calves after incubation periods of 24 and 26 days. Gut and salivary glands were collected from ticks on each day of the 23-day experiment and examined with light and electron microscopy. Colonies of A marginale were first observed in midgut epithelial cells on the sixth day of feeding on infected calves, with the highest density of colonies found in gut cells while ticks were between feeding periods. The first colonies contained 1 large dense organism that subsequently gave rise to many reticulated organisms. Initially, these smaller organisms were electron-lucent and then became electron-dense. On the fifth day after ticks were transferred to susceptible calves for feeding, A marginale colonies were found in muscle cells on the hemocoel side of the gut basement membrane. A final site for development of A marginale was the salivary glands. Colonies were first seen in acinar cells on the first day that ticks fed on susceptible calves, with the highest percentage of infected host cells observed on days 7 to 9 of that feeding. Organisms within these colonies were initially electron-lucent, but became electron-dense.

Preliminary studies of the development of Anaplasma marginale in salivary glands of adult, feeding Dermacentor andersoni ticks.

On each day of feeding on susceptible calves, salivary glands obtained from groups of adult ticks that transmitted Anaplasma marginale were examined for A marginale colonies by use of light microscopy and transmission electron microscopy. On day 8 of feeding, salivary glands were examined, using fluorescein-labeled antibody and methyl green-pyronine stain. Use of fluorescein-labeled antibody consistently revealed small numbers of fluorescent foci in salivary gland acinar cells obtained from ticks that had fed for 8 days. Colonies of A marginale were seen by transmission electron microscopy only in salivary gland acini of male ticks; these colonies could not be identified, using light microscopy, in companion 1-micron plastic sections stained with Mallory stain. Methyl green-pyronine stain, used commonly to detect theilerial parasites in tick salivary glands, did not differentiate A marginale from cytoplasmic inclusions normally found in salivary gland acinar cells.

Ultrastructure of anaplasmal inclusions (Pawhuska isolate) and their appendages in intact and hemolyzed erythrocytes and in complement-fixation antigen.

Hemolysis of parasitized erythrocytes augmented visualization of the anaplasmal inclusion, including its initial bodies, inclusion membrane, and inclusion appendage ("tail" or "band"). A dense attachment complex joined the appendage to the inclusion membrane (wall of the inclusion vacuole). The inclusion appendage consisted of tightly packed, interconnected laminae and assumed loop, dumbbell, and comet configurations described by other workers. The erythrocytic plasmalemma and the inclusion membrane had a thickness of 9.0 +/- 0.8 nm and similar structures. The initial bodies were covered by a thin inner organismic membrane (7.0 +/- 0.7 nm thick) attached to the organismic chromatin, an intermembranous matrix, and by an outer membranous sheath or pellicle (12.5 +/- 1.2 nm thick). Dense granular aggregates (24 to 40 nm in diameter) within chromatin clumps were the only structures in the initial body remotely similar to ribosomes, yet they were too large, were never free of chromatin, and appeared to disappear upon hemolysis. Complement-fixation antigen prepared by fractionation contained initial bodies, inclusion appendages, a few mitochondria, vesicularized membranes, and stromal debris. The preparatory treatment caused segregation of the organismic chromatin into independent dense particles 103 +/- 12 nm in diameter still bound by inner organismic membrane. Similar particles were seen also in the plasma and inclusion vacuoles of hemolyzed erythrocytes.

Demonstration of Anaplasma marginale Theiler in Dermacentor variabilis (Say) by ferritin-conjugated antibody technique.

Cattle were inoculated with a Virginia isolate of Anaplasma marginale Theiler and served as an infective source for laboratory-reared Dermacentor variabilis (Say) nymphs. Transstadial transmission of A marginale was demonstrated by feeding the newly molted adult ticks on susceptible cattle and by inoculation of gut homogenates collected from adult ticks at postattachment day 6. A similar gut homogenate from the same group of ticks was caused to react with A marginale-bovine antisera that had been conjugated with ferritin. The homogenate contained organisms similar to those observed in the gut of adult feeding ticks that were infected as nymphs and the labeling of the outer membrane confirmed that organisms observed were A marginale. A gut homogenate prepared from control ticks did not cause infection when inoculated into a susceptible cow and no organisms were demonstrated.

In vitro cultivation of Anaplasma marginale: growth pattern and morphologic appearance.

Anaplasma marginale propagated in vitro showed an increasing rate of replication in a sequence of three experiments. The changes in the percentage of parasitized erythrocytes and identification of the organism in culture were monitored by the Giemsa-staining and the direct fluorescent antibody techniques. The ultrastructure of the organism in culture also was determined. The percentage of parasitized erythrocytes increased more than three times in the first experiment during a period of 8 days, and about ten times in the second experiment during a period of 14 days. After 8 days of observation of the primary culture in the third experiment, the trend of growth was more rapid than in the first and second experiment. The viability of the organism was verified by inoculation of susceptible calves, using 13- and 33-day cultures of the first and the second experiments, respectively. The primary cultures were subcultured twice by dilution with normal bovine erythrocytes.