Serum cholesterol LDL and 90-day mortality in patients with intracerebral hemorrhage.

BACKGROUND AND PURPOSE: Prognostic significance of low-density lipoprotein cholesterol (LDL-C) in intracranial hemorrhage (ICH) is unclear. The objective of this study was to determine the association between LDL-C and mortality in ICH. METHODS: Consecutive patients (n=88) presenting with ICH were included in the study. Lipid profile was obtained during the first hours after admission. We analyzed the impact of LDL-C on 90-day mortality using the Hazard Rate (HR) crude, analysis crude for trend by Mantel-Haenszel Test, Multiple Cox Proportional Hazards model, and analysis of survival curves. Association between LDL-C and severity markers of ICH were explored using Spearman correlation coefficient. RESULTS: Low LDL-C levels were independently associated with death after intracranial hemorrhage (HR=3.07 (95% CI:1.04 to 9.02; P=0.042) in multivariable analysis after controlling for confounding factors. Analysis for trend showed a significant association (Xt=-2.144; P=0.032) by Mantel-Haenszel Test. Spearman analysis showed no correlation between LDL-C and variables that are markers of ICH severity: NIH score (r=-0.091; P=0.400), GCS score (r=0.136; P=0.207), ICH volume (r=0.140; P=0.192), and length of stay (r=-0.111; P=0.308). CONCLUSIONS: Low levels of LDL-C are independently associated with an increased risk of death in patients with brain hemorrhage. We have not found evidences that the levels of LDL-C can act as a biological marker of severity.

Primary midgut, salivary gland, and ovary cultures from Boophilus microplus.

Primary cell cultures from different tick organs are a valuable tool for host parasite research in the study of the protozoan Babesia sp., which infects different organs of the tick. In this work we describe the generation of midgut, salivary gland, and ovary primary cell cultures from dissections of Boophilus microplus. Midguts, salivary glands, and ovaries were dissected from B. microplus ticks on different days after bovine infestation; different enzymatic disaggregating protocols were tested in the presence of proteolytic enzymes, such as trypsin and collagenase type I and II, for tissue disaggregation and primary cell culture generation. The dissected tick organs obtained 18-20 days after bovine infestation showed a major cellular differentiation and were easier to identify by cellular morphology. The enzymatic disaggregation results showed that each tissue required a different proteolytic enzyme for optimal disaggregation; collagenase type I produced the most complete disaggregation for ovaries but not for midgut or salivary glands. Collagenase type II was effective for salivary glands but performed poorly on ovaries and midgets, and typsin was effective for midguts only. The midgut and ovary primary cell cultures were maintained for 4 weeks in optimal conditions after the cells were no longer viable. The salivary gland cell cultures were viable for 8 months.

Phylogenetic analysis of Mexican Babesia bovis isolates using msa and ssrRNA gene sequences.

Variable merozoite surface antigens of Babesia bovis are exposed glycoproteins having a role in erythrocyte invasion. Members of this gene family include msa-1 and msa-2 (msa-2c, msa-2a(1), msa-2a(2), and msa-2b). Small subunit ribosomal (ssr)RNA gene is subject to evolutive pressure and has been used in phylogenetic studies. To determine the phylogenetic relationship among B. bovis Mexican isolates using different genetic markers, PCR amplicons, corresponding to msa-1, msa-2c, msa-2b, and ssrRNA genes, were cloned and plasmids carrying the corresponding inserts were sequenced. Comparative analysis of nucleotide and deduced amino acid sequences revealed distinct degrees of variability and identity among the coding gene sequences obtained from 12 geographically different B. bovis isolates and a reference strain. Overall sequence identities of 47.7%, 72.3%, 87.7%, and 94% were determined for msa-1, msa-2b, msa-2c, and ssrRNA, respectively. A robust phylogenetic tree was obtained with msa-2b sequences. The phylogenetic analysis suggests that Mexican B. bovis isolates group in clades not concordant with the Mexican geography. However, the Mexican isolates group together in an American clade separated from the Australian clade. Sequence heterogeneity in msa-1, msa-2b, and msa-2c coding regions of Mexican B. bovis isolates present in different geographical regions can be a result of either differential evolutive pressure or cattle movement from commercial trade.

Enhancement of the Mexican bovine babesiosis vaccine efficacy by using Lactobacillus casei.

To evaluate the effect of Lactobacillus casei on the effectiveness of the Mexican bovine babesiosis mixed vaccine, 20 bovines were randomly allocated into four groups of five animals (I, II, III, and IV). At day -2 animals in groups I and II were inoculated with saline solution by intramuscular route (i.m.) and animals in groups III and IV were inoculated with L. casei. At day 0 bovines in groups I and III were inoculated i.m. with bovine normal erythrocytes and animals of groups II and IV were inoculated with the babesiosis vaccine. Twenty-four days later each bovine was challenged with Babesia bovis- and B. bigemina-infected erythrocytes. The average rectal temperature in groups I and III was higher (P < 0.05) than that in the vaccinated groups after challenge. The average packed cell volume was lower (P < 0.01) in the control groups than in the vaccinated groups. At day 10 after challenge, the average anti-Babesia antibody level was higher in group IV than in group II. At day 7 after vaccination, the percentage of bovines positive to gamma interferon, as determined by real-time PCR, was 20, 0, 40, and 80 for groups I, II, III, and IV, respectively. All animals in control groups (I and III) were treated against babesiosis to avoid their death because they showed signs of babesiosis. The results indicate that L. casei, inoculated 2 days before the inoculation of the Mexican bivalent bovine babesiosis vaccine, improves the vaccine's efficiency.

Expression analysis of heat shock protein 20 and rhoptry-associated protein 1a in sexual stages and kinetes of Babesia bigemina.

Heat shock protein 20 (HSP-20) and rhoptry-associated protein 1a (RAP-1a) are two proteins considered as candidates to be included in vaccines or diagnostics methods for the control of bovine babesiosis. It has been hypothesized that both genes have a basic function in the cellular physiology of erythrocyte-infecting stages; it is not known if they have a functional role in tick stages. The objective of this work was to analyze whether hsp-20 and rap-1a are expressed in sexual stages and kinetes of Babesia bigemina. Purified RNA from sexual stages and kinetes was analyzed by reverse transcriptase (RT)-PCR with specific primers for hsp-20 or rap-1a. Expression analysis was carried out using an indirect immunofluorescence test with specific antibodies against HSP-20 and RAP-1a. Results obtained by RT-PCR showed amplicons for hsp-20 and rap-1a in sexual stages and kinetes. Positive signals were also detected when sexual stages and kinetes were analyzed with specific antibodies for HSP-20 and RAP-1a. The results obtained here confirm the hypothesis that the genes hsp-20 and rap-1a from B. bigemina are expressed in sexual stages and kinetes and stress the importance of these proteins in the cellular physiology of tick stages.

msa-1 and msa-2c gene analysis and common epitopes assessment in Mexican Babesia bovis isolates.

Babesia bovis msa-1 and msa-2c genes belong to the variable merozoite surface antigen gene family. These genes code for antigenic proteins present on the merozoite surface (MSA) and are involved in the parasite invasion to the bovine erythrocyte. Previous studies carried out on MSA-1 have evidenced antigen allelic variation in B. bovis isolates from similar endemic regions, as well as in isolates from different geographic regions of the world (Argentina, Australia, Israel). Studies conducted on MSA-2c, however, have shown that this antigen is widely conserved on isolates from distinct geographic regions. In this study, it was hypothesized that MSA-1 and MSA-2c antigens would contain common epitopes despite the presence of nucleotide sequence differences found in 13 B. bovis isolates and strains collected in geographically distant regions of Mexico. Bioinformatics analysis of the primary structure from DNA fragments derived from PCR amplification, cloning, and sequencing of msa-1 and msa-2c genes from the 13 B. bovis populations revealed that the msa-1 gene product present in the various isolates tested is less conserved among isolates obtained within a similar geographic region in Mexico (51-99.7% sequence identity). Results obtained by immunoblot analysis of B. bovis protein extracts reacted with a monoclonal antibody to MSA-1 42-kDa antigen, conclusively showed cross-reactive common epitopes only in Mexican isolates having high sequence identity (>/=99%, eight isolates). Sequence analysis and multiple alignment of deduced MSA-2c demonstrated a high degree of sequence identity (90-100%) among the Mexican B. bovis isolates and strains. Immunoblot results using a polyclonal antibody to MSA-2c reacted against the protein extracts recognized conserved epitopes in at least nine of the B. bovis isolates. The results obtained in this study agree with those previously reported by other researchers and confirm that, based in sequence identity conservation in Mexican B. bovis isolates and strains so far collected and analyzed, MSA-2c represents an ideal antigen worth evaluating as a vaccine candidate.

Bovine babesiosis live vaccine production: use of gamma irradiation on the substrate.

Gamma irradiation on bovine serum and red blood cells (RBC) allows proliferation and growth of in vitro-cultured Babesia sp., and has potential application to inactivate contaminating viruses and bacteria from the substrate. Gamma irradiation with 25 kGy in a source of (60)Co was able to inactivate infectious bovine rinotracheitis (IBR) and bovine viral diarrhea (BVD) viruses in artificially contaminated serum; besides, bacteria were also eliminated. In vitro culture of Babesia bovis (B. bovis) in modified substrate, by adding irradiated serum with (60)Co at 25 kGy was propagated from 24-well culture plates to 225 cm(2) tissue culture flasks, and percentages of parasitized erythrocytes (PPE) from 2.4% to 8.8% were obtained. Infected RBC adapted to Irrad S were transferred to the irradiated substrate in vitro culture system, by using serum irradiated at 25 kGy and RBC from 10 to 70 Gy. The PPE ranged from 3.1 to 11. Culture of Babesia bigemina (B. bigemina) was established with Irrad S (25 kGy); its propagation was achieved in tissue culture flasks reaching PPE from 0.5 to 4.3 with no statistical difference (P > 0.05) when compared to the nonirradiated control culture (1.2-4.8). B. bigemina-infected RBCs were transferred to the modified culture system by adding irradiated serum and RBC (25 kGy and 70 Gy, respectively). PPE obtained in culture flasks were from 0.8 to 4.2. The results indicate that gamma irradiation is a suitable method to inactivate potential viral contamination and eliminate bacteria from bovine serum, to produce a live attenuated vaccine through the in vitro culture.

Babesia bigemina: sporozoite isolation from Boophilus microplus nymphs and initial immunomolecular characterization.

It has been hypothesized that babesial sporozoites express specific antigens that induce protective immunologic responses in cattle. However, they remain uncharacterized, partly for lack of research on the sporozoite stage of Babesia spp. This field suffers from complete knowledge of parasite development in the tick salivary gland; limited amounts of sporozoites from ticks, and a lack of protocols for induction and purification of sporozoites. In this work, Boophilus microplus larvae infected with B. bigemina were fed on susceptible cattle. Nymphs were collected and macerates were separated by a Percoll density gradient. Microscopic analysis of Giemsa-stained smears showed a larger number of sporozoites from nymphs fed for 9 days. Percoll-purified sporozoites were observed in large numbers in groups or individually and free of tick cells. RT-PCR analysis of total RNA extracted from purified sporozoites indicated transcription of the rhoptry associate protein 1 (rap-1) genes: rap-1a, rap-b, rap-1c, as well as the heat shock protein 20 (hsp-20) gene. Purified sporozoites were cultured in vitro analyzed for RAP-1a expression using an immunocytochemistry assay. Erythrocyte-attached sporozoites reacted with a specific RAP-1a monoclonal antibody. This is the first report of Babesia bigemina sporozoite antigens. Moreover, purified sporozoites will allow the characterization of stage-specific antigens involved in immunologic protection.

Babesia bigemina sexual stages are induced in vitro and are specifically recognized by antibodies in the midgut of infected Boophilus microplus ticks.

Babesia bigemina, a causative agent of bovine babesiosis, is transmitted from one bovine to another only by infected ticks. The life cycle of B. bigemina includes a sexual phase in the tick host; however, molecules from sexual stages of any Babesia species have not been characterized. This is the first report of the induction of sexual stages of any Babesia species in vitro, free of tick antigens. Intraerythrocytic parasites were cultured in vitro for 20h using an induction medium. Extraerythrocytic parasites were first seen 3h post induction; elongated stages with long projections appeared at 6h post induction and by 9h they paired and fused to form larger stages. Round zygotes appeared 20h post induction. Moreover, by using Percoll gradients, sexual stages were purified free of contaminating intraerythrocytic stages. Purified parasites were used to generate polyclonal antibodies, which specifically bound to antigens expressed in sexual stages induced in vitro, but not to antigens expressed in intraerythrocytic stages. Importantly, these antibodies specifically identified sexual stages from midguts of female Boophilus microplus ticks fed on infected cattle.