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Sera were collected from a minimum of 20 cattle aged nine to 36 months at each of 14 localities in five climatic zones of Sri Lanka. Sera were tested for antibodies to Babesia bovis and Anaplasma by an indirect fluorescent antibody test and a card agglutination test, respectively. Antibodies to B. bovis and Anaplasma were detected in all samples tested from each of 14 and 12 localities respectively. In general, prevalences were consistently high among localities below 1,200 m and lower and more variable in the hill country. Management practice rather than climate was considered to be the main factor influencing prevalences. Management is discussed in relation to the risk of disease outbreak.
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Blood and clotted blood samples from 3 calves with anaplasmosis were examined by light and electron microscopies before and after the calves were treated twice with alpha-ethoxyethylglyoxal dithiosemicarbazone (given IV). In the treated calves anaplasma bodies were swollen and irregularly shaped and had rough surfaces when viewed by light microscopy. Posttreatment electron microscopic examinations revealed swelling and vacuolation of initial bodies of anaplasma bodies, and ballooning of the vesicular membrane surrounding the anaplasma body. Other initial bodies were ameboid in shape this was or was not accompanied by rupture of the vesicular membrane of the anaplasma body. Necrotic, ameboid-shaped initial bodies also were seen. Some necrotic initial bodies were surrounded by complete vesicular membrane, and others were close to a broken, curled vesicular membrane.
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Two monoclonal antibodies (mAbs) for A. marginale were used to test the antigenic integrity of A. marginale grown in vitro in bovine erythrocytes co-cultured with endothelial cells. Both the mAbs reacted in the indirect immunofluorescent antibody test with A. marginale grown in vitro and also detected the antigens in Western immunoblots of SDS-PAGE separated antigens made from A. marginale infected erythrocytes from the cultures. Furthermore, active replication was evident as [35S]-methionine is incorporated by A. marginale present in the second passage of a culture maintained for six weeks as shown by immunoprecipitation of labeled antigens by the mAbs. This indicates that A. marginale grown in the in vitro culture system described previously [Waghela et al., Vet. Parasitol. 73 (1997) 43] maintain antigenic character, and with further development the system can be used for preparing immunogens or diagnostic antigens.
Antigenic characterization of A. marginale isolates has contributed to identifying the presence of common and restricts epitopes of major surface proteins (MSPs). The data may improve vaccine development to protect against A. marginale isolates from different regions. Brazilian A. marginale isolates were characterized antigenically by Western blot with monoclonal antibodies (MAbs) against MSPs and rabbit anti-MSP-4 from Florida strain. Six A. marginale isolates from MS, MG (AUFV1), SP, PR-L1, PR-HV, RS and Florida strain were tested with ANA22B1 to MSP-1a, AMR36A6 to MSP-1b, ANAF19E2 to MSP-2, AMG75C1 and AMG76B2 to MSP-3 and ANAF16C1 to MSP-5. ANA22B1 recognized MSP-1a epitope in all A. marginale isolates, and reacted with polypeptides of different size ranging 46-105kDa. MSP2 was not detected in MS and SP isolates by ANAF19E2, and only PR-L1 and MG (AUFV1) isolates reacted with MAbs which recognize MSP3 epitope. MSP4 and MSP5 were detected in all A. marginale isolates analyzed. The results revealed conservation of MSP-1a and MSP-5 epitopes among all Brazilian isolates, and showed antigenic variability to MSP-1b, MSP-2 and MSP-3 proteins, agreeing with recent data about the genetic diversity found in the polimorphic multigene family responsible for these proteins.
Serologic diagnosis of anaplasmosis is currently done by the complement-fixation, ELISA, and card agglutination tests. These tests have utilized A. marginale harvested from bovine erythrocytes as antigen which is often contaminated with erythrocyte stroma. We are currently testing A. marginale propagated in a Ixodes scapularis cell line as antigen for serologic tests. In this study, we report the use of the cell culture-derived A. marginale as antigen for development of a rapid, semi-automated latex agglutination test. Diluted serum and latex (polystyrene microspheres), sensitized with cell culture-derived A. marginale proteins, 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, 724/977 of the sera were positive for A. marginale antibodies 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.
Dermacentor andersoni nymphs were placed in stockinettes and allowed to feed on a splenectomized calf with experimentally induced anaplasmosis when the parasitemia was 3%-5%. Nymphs were selected on each of the 6 days of feeding and every 5 days from repletion through molting to the adult stage (25 days postrepletion); they were killed and midgut tissues were processed and examined by light and electron microscopies. No stages of A marginale were seen in tissues of feeding ticks. Visualization of individual components of gut contents was difficult owing to presence of the concentrated, electrondense blood meal containing hemoglobin. Inclusion appendages were observed in midgut tissues of nymphs at 5 and 10 days postrepletion, but not at 20 or 25 days. The morphology of the appendages was similar to that described for inclusion appendages commonly associated with anaplasmal inclusions in bovine erythrocytes. Some appendages were free in the lumen of the midgut and occurred either alone or with clusters of small vesicular particles. Occasionally, initial bodies like those generally found in bovine erythrocytes were seen with the appendage, but most of them were swollen and appeared to be degenerating. Frequently, inclusion appendages were observed attached to the luminal surface of the midgut cell membrane by a blunt, electron-dense attachment complex. The attachment of the appendage appeared to be extracellular, with the pointed end extending into the lumen. Often, small particles were observed immediately across the cell membrane from where the appendages were attached; the small particles appeared to be generated from the appendage itself and to have passed through the membrane of the midgut cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Neither imidocarb dipropionate (at 5 mg/kg body weight intramuscularly once or twice at a seven-day interval) nor oxytetracycline hydrochloride (at 20 mg/kg body weight intramuscularly once or twice at a seven-day interval) given to animals in the prepatent period of anaplasmosis, prevented the eventual onset of infection. Both compounds delayed the onset of detectable parasitaemia but this action was more noticeable with oxytetracycline. Oxytetracycline given twice significantly reduced the severity of the primary infection but the secondary infection was more severe. Neither compound showed prophylactic properties even though both are effective in extending the prepatent period and, in the case of oxytetracycline, reducing the severity of infection.
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The glycolytic process in non-infected and A. marginale-infected bovine erythrocytes was studied. The study included four enzymes: hexokinase, phosphofructokinase, pyruvic kinase and lactic dehydrogenase, and the intracellular concentrations of glucose, ATP and 2,3 diphosphoglyceric acid (DPG). The activities of phosphofructokinase and lactic dehydrogenase found in non-infected bovine erythrocytes were fifty times greater than the previously reported values. Glucose and DPG concentrations, hexokinase, pyruvic kinase and lactic dehydrogenase specific activities did not change significantly during experimental anaplasmosis. Phosphofructokinase activity in A. marginale-infected erythrocytes increased to values 300% of those found in intact bovine red cells. ATP concentration in infected erythrocytes decreased to values of 40% of those found in non-infected cells: from 1.60 +/- 0.40 to 0.70 +/- 0.08 mumole per gram of hemoglobin.
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