Babesia bovis-stimulated macrophages express interleukin-1beta, interleukin-12, tumor necrosis factor alpha, and nitric oxide and inhibit parasite replication in vitro

The tick-transmitted hemoparasite Babesia bovis causes an acute infection that results in persistence and immunity against challenge infection in cattle that control the initial parasitemia. Resolution of acute infection with this protozoal pathogen is believed to be dependent on products of activated macrophages (Mphi), including inflammatory cytokines and nitric oxide (NO) and its derivatives. B. bovis stimulates inducible nitric oxide synthase (iNOS) and production of NO in bovine Mphi, and chemical donors of NO inhibit the growth of B. bovis in vitro. However, the induction of inflammatory cytokines in Mphi by babesial parasites has not been described, and the antiparasitic activity of NO produced by B. bovis-stimulated Mphi has not been definitively demonstrated. We report that monocyte-derived Mphi activated by B. bovis expressed enhanced levels of inflammatory cytokines interleukin-1beta (IL-1beta), IL-12, and tumor necrosis factor alpha that are important for stimulating innate and acquired immunity against protozoal pathogens. Furthermore, a lipid fraction of B. bovis-infected erythrocytes stimulated iNOS expression and NO production by Mphi. Cocultures of Mphi and B. bovis-infected erythrocytes either in contact or physically separated resulted in reduced parasite viability. However, NO produced by bovine Mphi in response to B. bovis-infected erythrocytes was only partially responsible for parasite growth inhibition, suggesting that additional factors contribute to the inhibition of B. bovis replication. These findings demonstrate that B. bovis induces an innate immune response that is capable of controlling parasite replication and that could potentially result in host survival and parasite persistence.     

Induction of nucleoside transport sites into the host cell membrane of Babesia bovis infected erythrocytes

Normal bovine erythrocytes have negligible ability to transport adenosine and related nucleosides across their cell membrane. However, infection with the intraerythrocytic parasite Babesia bovis was found to induce a nucleoside permeation site into the host cell membrane. Transport experiments over periods of up to 30 s determined that the transport rate of 1 microM adenosine into the infected cell was 1.72 +/- 1.2 pmol incorporated (microliter cell water)-1s-1, a rate three times higher than for normal human erythrocytes. Incorporation studies over 6 h with labelled adenosine indicated that the purine moiety was incorporated into parasite nucleic acids. The mammalian nucleoside transport inhibitors, nitrobenzylthioinosine (NBMPR), nitrobenzylthioguanosine (NBTGR), dilazep and dipyridamole inhibited the induced nucleoside transport mechanism in the Babesia-infected erythrocytes, though at higher concentrations than those required to inhibit normal human erythrocyte transport. An ID50 value for NBMPR of 0.36 microM was determined. Phloretin and 5'-p-fluorosulphonyl benzoyl adenosine-HCl (5FSBA) were also shown to be inhibitory, with ID50 values of 0.11 and 0.18 microM, respectively, whilst phlorizin and verapamil at 1 microM had no effect. Binding studies with [3H]NBMPR indicated that high-affinity NBMPR binding sites could not be detected in either normal or B. bovis infected bovine erythrocytes. The results indicate that the induced nucleoside permeation site(s) in B. bovis infected erythrocytes has characteristics different from either human erythrocytes or erythrocytes infected with the malarial parasites Plasmodium falciparum or Plasmodium yoelii.     

The synthesis of lipids from [1-14C]acetate by isolated rat brain capillaries

Lipid biosynthesis was investigated in isolated cerebral microvessels obtained from adult Sprague-Dawley rats using [1-14C]acetate as precursor. All lipid classes were labelled by [1-14C]acetate. Neutral lipids incorporated about 50% of radiolabelled acetate, among which free fatty acids and triglycerids showed the highest level of incorporation. Moreover, about 4% of radioactivity was found in cholesterol fraction. In phospholipid fraction, phosphatidylcholine and phosphatidylethanolamine were the main radiolabelled phospholipids. [1-14C]acetate was also incorporated into sulphatides and cerebrosides. The presence of bovine serum albumin in incubation medium modified the percentage of incorporation in different lipid fractions.     

Effect of chlorpromazine on lipid metabolism in aortas from cholesterol-fed rabbits and normal rats, in vitro: Inhibition of sterol esterification and modification of phospholipid synthesis

Chlorpromazine (CPZ), a major tranquilizer, was found to be a potent inhibitor of acylCoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) in isolated arterial microsomes and in intact arterial tissue from the rat and cholesterol-fed rabbit in vitro. In isolated rabbit arterial microsomes, CPZ resulted in a concentration-dependent inhibition of ACAT with 50% inhibition of [1-¹⁴C]oleoylCoA incorporation into [¹⁴C]cholesteryl esters occurring at 0.1 mM CPZ. CPZ also effectively inhibited the incorporation of [¹⁴C]oleate into triglycerides without affecting incorporation into diglycerides. Additionally, CPZ altered the pattern of arterial phospholipids synthesized from [1-¹⁴C]oleate. Incorporation into phosphatidylcholine was depressed while incorporation into phosphatidylinositol was increased. Since diglyceride synthesis appreared to be unaffected by CPZ, a redirection of phosphatidic acid into the CDP-diglyceride pathway of glycerolipid synthesis does not adequately account for the effect of CPZ on arterial phospholipid and triglyceride synthesis in these experiments.     

rRNA-based method for sensitive detection of Babesia bigemina in bovine blood.

Three synthetic oligonucleotide probes complementary to unique regions of Babesia bigemina small-subunit rRNA were developed for detecting the parasite in bovine blood. These probes specifically detected a parasitemia of 2 x 10(-5)% by autoradiography in less than 24 h by using a 200-microliters sample of bovine blood. These probes did not bind to total RNA or genomic DNA isolated from another closely related species, Babesia bovis, or to bovine leukocyte RNA. This method detected B. bigemina infections in calves inoculated with as few as 1,000 infected erythrocytes from the second day onward for 16 days.     

Effect of endurance training on the phospholipid content of skeletal muscles in the rat

Only few data are available on the effect of training on phospholipid metabolism in skeletal muscles. The aim of the present study was to examine the effect of 6 weeks of endurance training on the content of particular phospholipid fractions and on the incorporation of blood-borne [¹⁴C]-palmitic acid into the phospholipids in different skeletal muscles (white and red sections of the gastrocnemius, the soleus and the diaphragm) of the rat. Lipids were extracted from the muscles and separated using thin-layer chromatography into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, cardiolipin and neutral lipids (this fraction being composed mostly of triacylglycerols). It was found that training did not affect the content of any phospholipid fraction in soleus muscle. It increased the content of sphingomyelin in white gastrocnemius muscle, cardiolipin and phosphatidylethanolamine in red gastrocnemius muscle and phosphatidylinositol in white gastrocnemius muscle and diaphragm. The total phospholipid content in red gastrocnemius muscle of the trained group was higher than in the control group. Training reduced the specific activity of sphingomyelin and cardiolipin in all muscles, phosphatidylcholine in soleus, red, and white gastrocnemius muscles, phosphatidylserine in all muscles, phosphatidylinositol in all except the soleus muscle, and phosphatidylethanolamine in hindleg muscles, but not in the diaphragm compared to the corresponding values in the sedentary group. It was concluded that endurance training affects skeletal muscle phospholipid content and the rate of incorporation of the blood-borne [¹⁴C]-palmitic acid into the phospholipid moieties.     

Cellular localization of Babesia bovis merozoite rhoptry-associated protein 1 and its erythrocyte-binding activity

The cellular localization of Babesia bovis rhoptry-associated protein 1 (RAP-1) and its erythrocyte-binding affinity were examined with anti-RAP-1 antibodies. In an indirect immunofluorescent antibody test, RAP-1 was detectable in all developmental stages of merozoites and in extracellular merozoites. In the early stage of merozoite development, RAP-1 appears as a dense accumulation, which later thins out and blankets the host cell cytoplasm, but retains a denser mass around newly formed parasite nuclei. The preferential accumulations of RAP-1 on the inner surface of a host cell membrane and bordering the parasite's outer surface were demonstrable by immunoelectron microscopy. An erythrocyte-binding assay with the lysate of merozoites demonstrated RAP-1 binding to both bovine and equine erythrocytes. Anti-RAP-1 monoclonal antibody 1C1 prevented the interaction of RAP-1 with bovine erythrocytes and significantly inhibited parasite proliferation in vitro. With the recombinant RAP-1, the addition of increasing concentrations of Ca(2+) accentuated its binding affinity with bovine erythrocytes. The present findings lend support to an earlier proposition of an erythrocytic binding role for RAP-1 expressed in B. bovis merozoites and, possibly, its involvement in the escape of newly formed merozoites from host cells.     

Phospholipid Composition of Purified Chlamydia trachomatis Mimics That of the Eucaryotic Host Cell

Chlamydia trachomatis is an obligate intracellular eubacterial parasite capable of infecting a wide range of eucaryotic host cells. Purified chlamydiae contain several lipids typically found in eucaryotes, and it has been established that eucaryotic lipids are transported from the host cell to the parasite. In this report, we examine the phospholipid composition of C. trachomatis purified from host cells grown under a variety of conditions in which the cellular phospholipid composition was altered. A mutant CHO cell line, with a thermolabile CDP-choline synthetase, was used to show that decreased host cell phosphatidylcholine levels had no significant effect on C. trachomatis growth. However, less phosphatidylcholine was transported to the parasite and purified elementary bodies contained decreased levels of phosphatidylcholine. Brefeldin A, fumonisin B₁, and exogenous sphingomyelinase were used to alter levels of host cell sphingomyelin. None of the agents had a significant effect on C. trachomatis replication. Treatment with fumonisin B₁ and exogenous sphingomyelinase resulted in decreased levels of host cell sphingomyelin. This had no effect on glycerophospholipid trafficking to chlamydiae; however, sphingomyelin trafficking was reduced and elementary bodies purified from treated cells had reduced sphingomyelin content. Exposure to brefeldin A, which had no adverse effect on chlamydia growth, resulted in an increase in cellular levels of sphingomyelin and a concomitant increase in the amount of sphingomyelin in purified chlamydiae. Under the experimental conditions used, brefeldin A treatment had only a small effect on sphingomyelin trafficking to the host cell surface or to C. trachomatis. Thus, the final phospholipid composition of purified C. trachomatis mimics that of the host cell in which it is grown.     

Lipid composition of metacestodes of Taenia taeniaeformis and lipid changes during growth

A lipid analysis was performed on developing metacestodes of Taenia taeniaeformis removed from the livers of rats at times varying from 3 to 35 weeks post infection. Lipid accounted for 7–21% of the dry weight of the parasites. The highest proportions were found at the earlier stages. The distribution was as follows; neutral lipid 27–45%; glycolipid 5–11%; and phospholipid 50–61%. The major neutral lipid was cholesterol, and minor neutral lipids were sterol esters, triglycerides, diglycerides and monoglycerides. Hydrocarbons were present throughout development, but in the highest amounts at the earlier stages. Five different glycolipids were found, all of which were identified as glycosphingolipids. An increase in the proportion of more complex glycolipids was noted as parasites grew older. Ten different phospholipids were identified, with the major components being phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Other phospholipids were: lysophosphatides, phosphatidylinositol, phosphatidic acid, diphosphatidylglycerol, sphingomyelin, and an unknown phospholipid component. Changes in the relative amounts of the two major phospholipids were found when the early and late stages were compared. Two lipids found throughout development were identified as glycosylated dolichol phosphates, and they comprised between 1 and 3% of the total phospholipid fraction. Nineteen fatty acids were detected, and the fatty acid distribution for each lipid class at each stage was determined. Seven major fatty acids were common to each. These were: hexadecanoic, octadecanoic, oleic, linoleic, arachidonic, docosanoic, and docosahexaenoic.     

Cytoadherence of Babesia bovis-Infected Erythrocytes to Bovine Brain Capillary Endothelial Cells Provides an In Vitro Model for Sequestration

Babesia bovis, an intraerythrocytic parasite of cattle, is sequestered in the host microvasculature, a behavior associated with cerebral and vascular complications of this disease. Despite the importance of this behavior to disease etiology, the underlying mechanisms have not yet been investigated. To study the components involved in sequestration, B. bovis parasites that induce adhesion of the infected erythrocytes (IRBCs) to bovine brain capillary endothelial cells (BBEC) in vitro were isolated. Two clonal lines, CD7(A+I+) and CE11(A+I-), were derived from a cytoadherent, monoclonal antibody 4D9.1G1-reactive parasite population. This antibody recognizes a variant, surface-exposed epitope of the variant erythrocyte surface antigen 1 (VESA1) of B. bovis IRBCs. Both clonal lines were cytoadhesive to BBEC and two other bovine endothelial cell lines but not to COS7 cells, FBK-4 cells, C32 melanoma cells, or bovine brain pericytes. By transmission electron microscopy, IRBCs were observed to bind to BBEC via the knobby protrusions on the IRBC surface, indicating involvement of components associated with these structures. Inhibition of protein export in intact, trypsinized IRBCs ablated both erythrocyte surface reexpression of parasite protein and cytoadhesion. IRBCs allowed to recover surface antigen expression regained the ability to bind endothelial cells, demonstrating that parasite protein export is required for cytoadhesion. We propose the use of this assay as an in vitro model to study the components involved in B. bovis cytoadherence and sequestration.     

Lipid metabolism in focal areas of normal-fed and cholesterol-fed pig aortas

The in vitro uptake and incorporation into various lipid fractions of [1-¹⁴C]oleic acid and [³²P]phosphate was investigated in focal areas of differing permeability in pig aortic intima and media identified in vivo by Evans blue dye uptake. In normal-fed pigs, no difference in the concentration of total cholesterol between blue and white areas of either intima or media was observed. Most of the oleic acid taken up by the aorta in the normal-fed pig was incorporated into phospholipid (primarily lecithin) and triglyceride with little incorporation into cholesteryl ester. The uptake and incorporation of the [1-¹⁴C]oleic acid into each of the lipid fractions studied was similar for blue and white areas of both intima and media. After 6 wk and 16 wk of cholesterol feeding, the concentration of cholesterol in the intima, but not in the media, of the blue areas were significantly greater than that in the corresponding white areas. In the 6-wk cholesterol-fed aortas, the uptake of ¹⁴C-labeled oleic acid and its incorporation into phospholipid, triglyceride and particularly into cholesteryl ester were all significantly increased in the blue areas of the intima compared with the corresponding white areas. This increase paralleled the increase in cholesterol concentration shown for these areas. No difference in uptake or incorporation of oleic acid into any of the lipid fractions studies occurred between blue and white areas from the 6-wk cholesterol-fed pig aortic media. After 16 wk of cholesterol feeding, there was no demonstrable difference between blue and white areas for either the intima or media with respect to the uptake of ¹⁴C-labeled oleic acid and its incorporation into lipid.With ³²P-labeled phosphate as precursor, no evidence for differing phospholipid synthesis in the blue and white areas of the normal-fed pig aortic intima or media could be obtained. When 6-wk cholesterol-fed pig aortic intima was studied, significant increases in sphingomyelin synthesis in blue compared to white areas were present. A trend towards increased incorporation into lecithin, phosphatidyl inositol and phosphatidyl ethanolamine and reduced incorporation of phosphatidyl inositol relative to lecithin was present in the blue compared with the white areas in the intima, but these differences were not statistically significant. No difference in [³²P]phosphate incorporation into phospholipid between the blue and white areas of the media was apparent for any of the phospholipid fractions.     

The effect of diabeton and metformin on the free-radical oxidation processes and the structural characteristics of the phospholipid composition of the erythrocyte membranes in non-insulin-dependent diabetes mellitus

Newly detected noninsulin--dependent diabetes mellitus is shown to have changes in phospholipid structure of erythrocytic membranes: a fall in total phospholipids, sphingomyelin, phosphatidylcholine, phosphatidic acid, a trend to increasing lysophosphatidylcholine. The shifts go in parallel with lipid peroxidation activation. Carbohydrate metabolism compensation is not enough to eradicate the shifts. The process is also dependent on the properties of a sugar-reducing drug. Diabeton is recommended as beneficial in this respect, while metformin is efficient in combinations.     

Renewal of the membrane complex of Schistosoma mansoni is closely associated with lipid metabolism

Metabolic pathways leading to lipid biosynthesis in four different developmental stages of Schistosoma mansoni were explored and quantified by incubation in the presence of labeled precursors in a chemically defined medium. At the schistosomulum stage and in male, female, or paired worms, glycerol and oleate incorporation into neutral lipids, mainly in the form of triacylglycerols, was greater than into phospholipids, whereas in 11-and 15-day-old worms, synthesis mainly led to phospholipids. Incorporation into phospholipids was recovered largely in phosphatidylcholine, and distribution into other phospholipids depended on the developmental stage. Incorporation of choline and ethanolamine into their respective phospholipids represented up to 15% of the parasitic phospholipid content. The formation of phosphatidylcholine by phosphatidylethanolamine methylation occurred mainly in the immature parasitic stages. Inositol incorporation was also measurable, whereas [14C]serine incorporation was low or undetectable. Addition of 1-palmitoyl-2-[14C]oleyl phosphatidylcholine revealed a very high uptake of this phospholipid by the immature stages but further metabolism was not detectable. In contrast, adult S. mansoni were completely unable to take up or absorb this exogenous phospholipid. The most striking aspect of this study was the relatively high metabolic activity in 11-day-old worms and the lower but sustained activity on day 15 and at the schistosomulum stage. By comparison, biosynthetic activity in adult S. mansoni, on which research studies have been focused until now, was very low. We also discuss the participation of lipid metabolism in the constant renewal of the membrane complex which is essential to parasitism by S. mansoni.     

Phospholipid metabolism of cultured Trichomonas vaginalis and Tritrichomonas foetus

Trichomonas vaginalis and Tritrichomonas foetus grown in a fetal calf serum-based culture medium were exposed to radiolabeled phospholipids and lipid precursors to determine the extent to which these organisms can incorporate complex lipids and/or de novo synthesize their major membrane phosphoglycerides. Phosphatidylethanolamine and phosphatidylcholine were the dominant phospholipids (40-50% of extractable phospholipids), with acidic lipids, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and O-acylphosphatidylglycerol accounting for the remaining phosphoglycerides. T. vaginalis was rich in sphingomyelin while T. foetus lacks significant amounts of this lipid. Incubation with [32P]orthophosphate resulted in only modest incorporation into extractable phospholipids; the most striking observation being the failure to label choline-containing lipids (phosphatidylcholine, lysophosphatidylcholine and sphingomyelin). Phosphatidylethanolamine was heavily labeled with modest labeling observed in the acidic phosphoglycerides. [U-14C]Glucose failed to label choline-containing lipids in T. foetus but did so in T. vaginalis, with phosphatidylethanolamine again being heavily labeled. Choline, phosphorylcholine, ethanolamine, serine, inositol, glycerol and methionine were incorporated poorly or failed to label the expected phosphoglycerides in either of the trichomonads, demonstrating an impairment in synthesis. Intact phosphoglycerides, labeled in the fatty acyl groups, labeled most phospholipids indicating that turnover of membrane lipids can occur with respect to the acyl component of the phospholipids. Fluorescent probes attached to phosphoglyceride molecules support observations seen with radiolabeled phosphoglycerides. Though trichomonads are able to transacylate phosphoglycerides, it is evident that the trichomonads lack a variety of enzymatic activities necessary for de novo synthesis of complex phosphoglycerides and must rely on environmental sources to supply them.     

Investigation of the nature of potential phosphorylcholine donors for filarial nematode glycoconjugates

Filarial nematodes produce proteins containing phosphorylcholine (PC) covalently attached to N-linked glycans. Our previous work has suggested that transfer of PC might be dependent on a metabolite of the Kennedy pathway of phospholipid biosynthesis. In this study we have investigated whether the end product of this pathway, phosphatidylcholine, and in addition, sphingomyelin, could act as PC donors. Pulse-chase experiments employing [3H]choline as radiolabel, ruled out sphingomyelin, as the Acanthocheilonema viteae PC-containing protein, ES-62, was radiolabelled 20-30 min prior to the lipid. Phosphatidylcholine however was labelled immediately before ES-62 increasing the possibility that it could act as donor. This was further investigated by radiolabelling phosphatidylcholine synthesised via an alternative pathway such that other metabolites in the Kennedy pathway were not labelled. Specifically, we labelled the choline component of phosphatidylcholine using both [3H]serine and [3H]S-adenosyl methionine (SAM). Incubation of worms with [3H]serine failed to result in labelling of the PC component of ES-62 whereas the presence of [3H]SAM in the medium led to labelling of ES-62 but only 24 h after labelling of phosphatidylcholine. As ES-62 is labelled within minutes of phosphatidylcholine when employing [3H]choline as radiolabel, this suggests that labelling of ES-62 when using SAM is not due to direct transfer of PC from phosphatidylcholine. It is therefore concluded that neither sphingomyelin nor phosphatidylcholine act as PC donors for filarial nematode glycoproteins. The analysis of PC-containing metabolites and products from A. viteae additionally revealed the presence of PC-substituted glycolipids that were also radiolabelled by the use of [14C]choline. The kinetics of radiolabelling however differed from that observed in the case of ES-62, sphingomyelin and phosphatidylcholine in so far as labelled glycolipids were first detectable hours rather than minutes after addition of [14C]choline.     

cDNA clone encoding a high molecular weight antigen of Babesia bovis

An expression library was constructed by inserting cDNA copied from mRNA of the blood stages of Babesia bovis isolate KA into bacteriophage lambda gt11-amp3. An antigen-positive cDNA clone detected by screening the library with antibodies from cattle vaccinated with the KA isolate was shown to encode part of a high-molecular weight polypeptide antigen of B. bovis. This molecule was a dominant immunogen and was found by immunofluorescence to be within the parasite in infected erythrocytes.     

Effect of <Emphasis Type="Italic">Rhaponticum carthamoides</Emphasis> extract on structural and metabolic parameters of erythrocytes in rats with cerebral ischemia

Cerebral ischemia in rats was accompanied by an increase in erythrocyte degradation, which results from changes in lipid composition of their membranes. The content of lipids and phospholipid fraction (phosphatidylcholine, sphingomyelin, and phosphatidylserine) decreased, while the relative content of lysophospholipids increased in erythrocyte membranes. The course of treatment with Rhaponticum carthamoides extract (150 mg/kg perorally, 5 days) contributed to an increase in the contents of total lipids and phospholipids (primarily of sphingomyelin and phosphatidylserine) and decrease in the ratio of lysophospholipids in the erythrocyte membrane of rats with cerebral ischemia. Morphological characteristics of erythrocytes returned to normal, which manifested in an increase in the number of discocytes and decrease in the count of degenerated cells. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 146, No. 7, pp. 50–53, July, 2008     

Serum containing tumor necrosis factor is cytotoxic for the human malaria parasite Plasmodium falciparum.

Sera (BCG-lipopolysaccharide [LPS] serum) were obtained from mice infected with Mycobacterium bovis BCG 2 h after intravenous administration of bacterial endotoxin (LPS). Varying concentrations of sera were added to cultures of Plasmodium falciparum-infected human erythrocytes; parasite viability was assessed by hypoxanthine incorporation after 4 days in culture. At concentrations of 1 to 3%, cultures treated with BCG-LPS serum showed a two- to threefold increase in hypoxanthine incorporation; at higher concentrations (4 to 8%), hypoxanthine incorporation fell to 2 to 5% of that in control cultures. Concurrent assays with control sera (from untreated mice or mice treated with BCG or LPS alone) caused some stimulation but no inhibition at up to 8% concentration. Examination of cultures treated with BCG-LPS serum showed morphological, deterioration of parasites within erythrocytes. The presence of tumor necrosis factor in the BCG-LPS serum was confirmed by using a standard L-cell cytotoxicity assay. In addition, rabbit antiserum against partially purified tumor necrosis factor protected intraerythrocytic forms of P. falciparum from the toxic effects of BCG-LPS serum. These data suggest that the factor in BCG-LPS serum that is toxic to P. falciparum in human erythrocytes is antigenically similar or identical to tumor necrosis factor. This nonantibody mediator of killing may play a role in human malaria.     

Lipid components of the hydrocarbon assimilating yeast Candida lipolytica (strain 10)

The utilization of n-hexadecane by Candida lipolytica (strain 10) was studied with respect to the lipid content, phospholipid and fatty acid profiles resulting at various growth times. Thin layer chromatography of the lipid extracts showed quantitative changes in the different lipid classes. The phospholipid fraction obtained at each growth time was separated into 8 classes: lysophosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, glycophospholipid, phosphatidylglycerol, cardiolipin, and phosphatidic acid. Differences in the percentage fatty acid composition of the lipid extracts were observed at various stages of growth. The cellular fatty acids included palmitic, palmitoleic (35–52%), stearic, oleic, linoleic (26–39%), and pentadecanoic (2–12%) as major components. This indicates that fatty acid(s) of the same length as that of the substrate was the most abundant component, thus showing intact incorporation mechanism. Fatty acids having longer chain lengths were also formed in substantial amounts indicating C₂ addition and β-oxidation of the fatty acids formed in the yeast.     

Detection of culture-derived Babesia bovis exoantigen using a two-site enzyme immunoassay.

Soluble exoantigens in the supernatants of Babesia bovis cultures have been shown to be efficient immunogens against bovine babesiosis. We used a two-site enzyme immunoassay to monitor the release of these antigens during in vitro cultivation. Bovine immunoglobulin G was isolated from serum of an adult cow previously immunized with culture-derived B. bovis exoantigens and challenged via needle with virulent parasites. The specific immunoglobulin G was used as a capture antibody and as an enzyme-conjugated recognizing antibody. The optimal protein concentration of capture antibody was 10 micrograms/ml. The 24-h cultures showed the greatest antigen concentration. The test was sensitive for detection of differences in species-specific antigenic activity among B. bovis isolates, for determining loss of antigenicity during storage and formalinization, and for monitoring the kinetics of exoantigen release during in vitro cultivation. Antigens cross-reactive with the other major Babesia species of cattle, Babesia bigemina, were also detected with this assay. The high specificity, sensitivity, and reproducibility of this technique should facilitate detection and quantitation of Babesia antigens during purification and in standardization of candidate immunogens.