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PubMed1987

Infectivity of three Anaplasma marginale isolates for Dermacentor andersoni.

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.

Anaplasma↗

PubMed1985

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

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

Anaplasma↗

PubMed1986

Identification of antigens of two isolates of Anaplasma marginale, using a western blot technique.

Antigens of the Illinois (IAM) and Florida (FAM) isolates of Anaplasma marginale were analyzed, using the western blot technique and antiserum from A marginale-infected calves. Crude antigens were prepared from the parasitemic blood of each. Antiserum was collected after the primary and recrudescent parasitemias. Antigens were separated, using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Antigens were then transferred onto nitrocellulose membranes and exposed to test sera. Antibodies attached to the membrane-bound antigens were detected, using an avidin/biotin peroxidase assay and biotinylated rabbit anti-goat immunoglobulin G. Antigens detected were of a high molecular weight group (108 to 91 kilodaltons [kd]) or of a low molecular weight group (47 to 27 kd). The IAM antigens were 100 kd, 96 kd, 47 kd, 38 to 43 kd, and 27 kd; these antigens were detected, using anti-IAM and anti-FAM antibodies, but the anti-FAM antibodies had a strong reaction to only the 100-kd and 38- to 43-kd antigens of IAM. The FAM antigens were 108 kd, 91 kd, 47 kd, 38 to 43 kd, and 27 kd; these antigens were detected, using anti-FAM antibodies and, except the 91 kd antigen, anti-IAM antibodies. Because the 91-kd antigen was detected only in the FAM antigen and detected only by sera from FAM-infected calves, this isolate-specific antigen may be associated with the ability of FAM to induce disease in an IAM-immune animal. Sheep anti-A ovis antibodies reacted only to the 38- to 43-kd antigens of each isolate, indicating that these antigens may be genus-specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Anaplasma↗

PubMed1979

Characterization of the inclusion limiting membrane of anaplasma marginale by immunoferritin labeling.

Purified anti-erythrocytic membrane antibody (PAMA) was prepared from rabbit anti-bovine erythrocyte serum by an adsorption and elution technique, utilizing bovine erythrocytes. Lysed and washed anaplasma-infected erythrocytes were incubated with PAMA or control reagents. Specimens were then subjected to immunoferritin labeling with ferritin antiglobulin conjugate. Upon examination by electron microscopy, specimens incubated with PAMA showed heavy ferritin labeling of erythrocytic membranes and also the limiting membranes of anaplasmal inclusions. Anaplasmal initial bodies freed from their inclusion membranes were not labeled. Negative control specimens, incubated with normal rabbit serum or PAMA which had been absorbed with erythrocytes, did not show specific ferritin labeling. Intact bovine erythrocytes, which were used as a positive control of anti-bovine erythrocytic membrane specificity, were heavily ferritin-labeled. Avian erythrocytes, a negative control of specificity, remained unlabeled. The results of this study indicate that the limiting membrane of the anaplasmal inclusion is derived from the erythrocytic membrane.

Anaplasma↗

PubMed1979

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

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

Anaplasma↗

PubMed1981

Staining characteristics of colonies of Anaplasma marginale Theiler in Dermacentor andersoni Stiles.

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.

Anaplasma↗

PubMed1984

A rapid staining procedure for Anaplasma marginale in bovine erythrocytes.

A commercial product that stains cells in less than 1 minute was compared with the Giemsa and the Wright-Giemsa methods for detection of the intraerythrocytic parasite, Anaplasma marginale, in bovine blood smears. The total number of A marginale-infected erythrocytes detected in 5 microscope fields of 18 different samples was determined for each staining procedure. In samples where less than 2% to greater than 25% of the RBC were infected, statistical analysis of the data indicated that the rapid staining method was superior to the Giemsa and Wright-Giemsa methods with regard to determination of the percentage of bovine erythrocytes infected with A marginale.

Anaplasma↗

PubMed1982

Immunocytochemical labeling of Anaplasma marginale Theiler in Dermacentor andersoni Stiles with peroxidase- antiperoxidase technique.

Colonies of Anaplasma marginale in midgut epithelial cells of adult ticks that had been infected as nymphs were specifically labeled, using the unlabeled antibody peroxidase-antiperoxidase method of immunocytochemistry. Visual comparison of infected and control tissue sections with the electron microscope demonstrated deposition of ring-like peroxidase-antiperoxidase complexes over organisms within the colonies. The intensity of labeling differed among organisms within a single colony, possibly as a result of varying antigenicity. The labeling observed on organisms in the colonies was similar to that seen on anaplasmal initial bodies in inclusions of infected bovine erythrocytes examined concurrently.

Anaplasma↗

PubMed1983

Demonstration of Anaplasma marginale in hemolymph of Dermacentor andersoni by animal inoculation and by fluorescent-antibody technique.

Hemolymph was collected from adult Dermacentor andersoni Stiles that had been infected with Anaplasma marginale Theiler as nymphs. Before hemolymph was collected, the adult ticks were either incubated and unfed at 37 C for 2.5 days or fed for 6 days on sheep. Hemolymph collected from groups of 100 ticks was inoculated into susceptible splenectomized calves. Smears of hemolymph from the same groups of ticks were prepared for examination by fluorescent antibody technique. Hemolymph from incubated ticks caused anaplasmosis in 2 of 4 trials, and hemolymph from feeding ticks caused anaplasmosis in 4 of 4 trials. Moderately fluorescing bodies were demonstrated in some hemocytes from incubated ticks, whereas hemocytes from feeding ticks contained numerous clusters of brightly fluorescing bodies. Fluorescing bodies were not observed in hemocytes from control ticks.

Anaplasma↗

PubMed1983

Demonstration of colonies of Anaplasma marginale in the midgut of Rhipicephalus simus.

Rhipicephalus simus nymphs were allowed to feed on a cow experimentally infected with the BW-strain of Anaplasma marginale from Republic of South Africa, and they were studied as adults. Colonies were demonstrated by light microscopy in midgut epithelial cells of adult ticks that were unfed (as adults), incubated, or prefed for 72 hours on a cow. The colonies occurred in 5 different morphologic types (1 to 5) that were similar to those described previously for a Virginia isolate of A marginale in Dermacentor andersoni. The colony density (number of colonies/0.001 mm2 midgut tissue examined) ranged from 0 to 2.0 and was highest in unfed ticks that were not incubated (mean 0.566). Colonies observed by light microscopy were sectioned for study with the electron microscope. The colonies contained both electron-dense forms and reticulated forms. The organisms in type 2 and 3 colonies appeared to be attached to one another, and those in type 4 and 5 colonies occurred separately. Small particles were seen within the limiting membrane of some organisms. A few colonies contained a dense matrix and were surrounded by many small electron-dense particles.

Anaplasma↗

PubMed1983

Influence of parasitemia level at feeding on development of Anaplasma marginale Theiler in Dermacentor andersoni Stiles.

Four splenectomized dairy calves were inoculated with a Virginia isolate of Anaplasma marginale Theiler and served as an infective source (donor) for laboratory-reared Dermacentor andersoni Stiles nymphs. Two donor calves developed higher parasitemias during tick feeding than did the 2 other donor calves. One month after molting, adult ticks were incubated at 37 C for 2.5 days to stimulate development of colonies of A marginale, and homogenates of gut were made from ticks fed on each donor calf. Eight susceptible, splenectomized dairy calves (2 per donor calf) were each inoculated IV with the gut homogenates collected from 50 adult ticks and were monitored for patent A marginale infection. Gut tissues from these same groups of ticks were processed for histologic studies. Homogenates from ticks infected on donor calves with lower parasitemias caused infections with longer prepatent periods (av, 43.25 days) than did similar homogenates made from ticks infected by feeding on donor calves with higher parasitemias (av, 31.0 days). Also, sections of gut tissues from ticks infected at the lower parasitemias contained fewer (av, 0.65) colonies/0.001 mm2 of tissue examined than did those from ticks infected at higher parasitemias (av, 6.31).

Anaplasma↗

PubMed1983

Effects and use of a modified live Anaplasma marginale vaccine in beef heifers in California.

Eighty-one 11-month-old, nonpregnant, Anaplasma marginale-seronegative beef heifers were allotted to 2 groups for evaluation of a modified live A marginale vaccine (n = 50 for vaccinated group and n = 31 for nonvaccinated group). The vaccine induced a mild form of anaplasmosis, as evidenced by a significant (P less than 0.01) decrease in the packed cell volume (PCV) between days 31 and 46 after vaccination. The lowest PCV was 11%, and 3 heifers had a PCV less than 20%. Slight lethargy was evident in some of the vaccinated heifers between days 30 and 45 after vaccination. All vaccinated heifers became seropositive to A marginale, as measured by the complement fixation test and the card test on days 35 and 42 after vaccination, respectively. All nonvaccinated heifers remained seronegative.

Anaplasma↗

PubMed1980

Demonstration of Anaplasma marginale Theiler in ticks by tick transmission, animal inoculation, and fluorescent antibody studies.

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. Transstadial transmission of A marginale was demonstrated by feeding the newly molted adult ticks of both species on susceptible cattle and by inoculation of gut and salivary gland homogenates collected from adult ticks at postattachment days 5 and 7. Oral secretions collected from the same group of ticks and inoculated into a susceptible cow did not cause infection. Fluorescent antibody studies of infected tick gut and salivary glands were positive, although smears of tick oral secretions were negative for fluorescence.

Anaplasma↗

PubMed1982

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.

Anaplasma↗

PubMed1982

Preparation and laboratory testing of a frozen vaccine containing Babesia bovis, Babesia bigemina and Anaplasma centrale.

Procedures used to prepare and test frozen vaccine against bovine tick fever are described. Blood from splenectomised calves infected separately with Babesia bovis, Babesia bigemina and Anaplasma centrale was diluted in the ratio of 3:1 with 8 M dimethyl sulphoxide in phosphate buffered saline and cooled at rates between 5 degrees C and 110 degrees C per minute to -196 degrees C. After varying periods of storage up to 369 days, blood was thawed by immersing containers in a 40 degrees C water bath and its infectivity tested in a total of 150 nonsplenectomised cattle. Subcutaneous inoculation of the cryopreserved blood, and 10-fold and 50-fold dilutions prepared from it, showed that high infectivity of all three parasites was retained during storage. The use of frozen vaccine exported from Australia to Trinidad and Tobago is briefly discussed.

Anaplasma↗

PubMed1982

Influence of increased temperature on Anaplasma marginale Theiler in the gut of Dermacentor andersoni stiles.

Three splenectomized dairy calves were inoculated with a Virginia isolate of Anaplasma marginale Theiler and served as an infective source for laboratory-reared Dermacentor andersoni Stiles nymphs. One month after molting, groups of adult ticks were incubated at 37 C for 0, 1.5, 2.5, and 7 days. Gut homogenates were made from ticks representing each incubation period. Twenty-four susceptible, splenectomized dairy calves were each inoculated IV with a gut homogenate extracted from 50 adult ticks and monitored for patent A marginale infection. The prepatent periods were determined and used as a measure of infectivity. Gut homogenates made from ticks that had been incubated for 2.5 days produced infections with the shortest prepatent periods, an average of 28.5 days among 3 trials.

Anaplasma↗

PubMed1982

Isolation of an in vitro-produced soluble Anaplasma-albumin complex.

A soluble Anaplasma-albumin complex was isolated from supernatants of A marginale cultures by affinity chromatography. Holstein calves inoculated with this fraction produced anti-A marginale antibodies detected in the indirect fluorescent antibody test at titers similar to those found in cattle immunized with unfractionated culture supernatant. Titers of both groups peaked slightly above 1:2,000 on postinoculation day 46.

Anaplasma↗

PubMed1982

Elimination of naturally acquired chronic Anaplasma marginale infections with a long-acting oxytetracycline injectable.

Four injections of a long-acting oxytetracycline (20 mg/kg) were administered at 3-day intervals to 14 naturally infected anaplasmosis carrier cattle. Individual anaplasma serology titers were evaluated, using complement-fixation and rapid card-agglutination tests at frequent intervals during a 180-day posttreatment period. Serologic results indicated fluctuating complement-fixation titers during the trial period. However, at trial termination, 12 of 14 animals were negative by complement-fixation testing and all animals were card-test negative. Eight splenectomized calves given 10 ml of blood from the medicated cows at their posttreatment day 150 failed to manifest any clinical, hematologic, or serologic signs of anaplasmosis during 56 days. Susceptibility to anaplasmosis challenge exposure was demonstrated by positive clinical and serotest reactions when 3 of the recipient calves were inoculated with blood from a parasitemic carrier calf.

Agglutination Tests↗