Serum monitoring of IgG and IgM Babesia bigemina (Haemosporidia: Babesiidae) antibodies in calves from the Mexican tropics

Antibody dynamics (IgG and IgM) against Babesia bigemina was studied on 41 under 15 days of age from three ranches (R1, R2 and R3) in Yucatan, Mexico. Blood samples were collected every 30 days, for eight months. Sera were tested by the indirect fluorescent antibody method to detect IgG and IgM. Overall IgM seroprevalence during the calves first eight months of life was 17.1% without relation to age. Overall IgG seroprevalence was 66.8%, increasing with age. Seroprevalence in R1, R2 and R3 were 87.5%, 77.1% and 31.8% respectively. Ranches 1 and 2 were in enzootic stability. In Yucatan, the modification of management factors in ranches with enzootic instability, could increase the risk of clinical babesiosis. Cattle mobilization from ranches with enzootic instability must be strictly controlled.     

Ultrastructure of Babesia WA1 (Apicomplexa: Piroplasma) during infection of erythrocytes in a hamster model

Babesia Washington-1 (WA1) is a newly identified intraerythrocyte infectious agent of human babesiosis in the western United States. The purpose of the present study is to describe the ultrastructural changes in affected erythrocytes during the infectious process in a susceptible animal model, the golden Syrian hamster. Two, 1-mo-old female hamsters were inoculated intraperitoneally (i.p.) with 1.8 x 10(9) Babesia WA1-infected erythrocytes originally isolated from a human case and serially passaged in hamsters. Saphenous vein blood samples (20 microl) were collected at 0, 24, 36, 48, 60, 72, 84, and 96 hr postinoculation (PI). Parasitemia was determined at each time interval by quick staining of blood smears showing 0, 2.5, 5, 10, 12.5, 22.5, 70, and almost 100% parasitemic erythrocytes at the corresponding PI time interval, respectively. Animals showed weakness and dehydration 72 hr PI inoculation, and were killed by 96 hr PI. Selected blood samples from 0, 24, 48, 72, and 96 hr were fixed in cacodylate buffer, dehydrated in ethanol gradients, resin embedded, and then thin sectioned and stained with uranyl acetate and lead citrate for transmission electron microscopy or gold-coated for scanning electron microscopy (SEM). Shape and surface membrane changes in erythrocytes were demonstrated by SEM and were more evident at 72 and 96 hr PI. Infected erythrocytes underwent changes in shape 24 hr PI, from few protrusions to several perforations, some of them resembling a "swiss cheese" appearance 96 hr PI. Several erythrocytes had irregular surface membranes and Babesia WA1 organisms were seen at different stages of development within erythrocytes, from single trophozoites to several merozoites (young trophozoites), some of them dividing to form typical tetrads. In general, Babesia WAI induced severe morphological changes in the erythrocytes, and these changes were more evident in almost all infected cells 96 hr PI.     

Development of an in vitro microtest to assess drug susceptibility of Babesia bovis and Babesia bigemina.

Continuous cultivation of the bovine hemoparasites Babesia bovis and Babesia bigemina was developed as an in vitro microtest to assess parasite susceptibility to babesicidal compounds. Reproducibility of parasite multiplication rates was independent of culture size, making it possible to use a microscale of 100 microliters for each test sample. Inhibitory concentrations (IC50s) of a commonly used babesicide, quinuronium sulfate, evaluated by this in vitro method were found to be 5 x 10(-8) g/ml for B. bovis and 2 x 10(-9) g/ml for B. bigemina.     

Nilgai antelope in northern Mexico as a possible carrier for cattle fever ticks and Babesia bovis and Babesia bigemina

Of 20 blood samples from nilgais from México, five were polymerase chain reaction-positive for Babesia bigemina and one for Babesia bovis. Positive samples had the expected 170 (B. bigemina) and 291 (B. bovis) base pairs and were identical to Gen-Bank B. bigemina accession S45366 and B. bovis M38218.     

Description of Babesia duncani n.sp. (Apicomplexa: Babesiidae) from humans and its differentiation from other piroplasms

The morphologic, ultrastructural and genotypic characteristics of Babesia duncani n.sp. are described based on the characterization of two isolates (WA1, CA5) obtained from infected human patients in Washington and California. The intraerythrocytic stages of the parasite are morphologically indistinguishable from Babesia microti, which is the most commonly identified cause of human babesiosis in the USA. Intraerythrocytic trophozoites of B. duncani n.sp. are round to oval, with some piriform, ring and ameboid forms. Division occurs by intraerythrocytic schizogony, which results in the formation of merozoites in tetrads (syn. Maltese cross or quadruplet forms). The ultrastructural features of trophozoites and merozoites are similar to those described for B. microti and Theileria spp. However, intralymphocytic schizont stages characteristic of Theileria spp. have not been observed in infected humans. In phylogenetic analyses based on sequence data for the complete18S ribosomal RNA gene, B. duncani n.sp. lies in a distinct clade that includes isolates from humans, dogs and wildlife in the western United States but separate from Babesia sensu stricto, Theileria spp. and B. microti. ITS2 sequence analysis of the B. duncani n.sp. isolates (WA1, CA5) show that they are phylogenetically indistinguishable from each other and from two other human B. duncani-type parasites (CA6, WA2 clone1) but distinct from other Babesia and Theileria species sequenced. This analysis provides robust molecular support that the B. duncani n.sp. isolates are monophyletic and the same species. The morphologic characteristics together with the phylogenetic analysis of two genetic loci support the assertion that B. duncani n.sp. is a distinct species from other known Babesia spp. for which morphologic and sequence information are available.     

Babesia bigemina: quantitation of infection in nymphal and adult Boophilus microplus using a DNA probe.

Candidates for a subunit vaccine against bovine babesiosis include surface proteins of infective forms found in the salivary glands of tick vectors. However, low numbers of infective forms are present within ticks and hinder analysis of this stage. To solve this problem, conditions which yield high numbers of infective forms were investigated with the use of a Babesia bigemina-specific DNA probe. DNA from progeny of female Boophilus microplus infected with B. bigemina was hybridized to probe DNA to detect and quantitate infection. There was no difference in the prevalence of infection in progeny of three strains of Bo. microplus. However, within a strain, prevalence could be increased to 30% by combining selection of progeny from heavily (3+) infected female ticks and selection of eggs laid 120 hr postengorgement. Quantitation of infective forms within pooled salivary gland preparations of 10 infected nymphal and adult Bo. microplus demonstrated that Day 9 and 10 nymphal ticks contained the highest numbers of parasites and represented approximately 10(6) infective forms. This number of infective forms is suitable for isolation and further characterization.     

In vitro culture and fructan production by Vernonia herbacea (Asteraceae)

Vernonia herbacea is a native species from the Brazilian Cerrado that accumulates about 80 % of inulin-type fructans in the underground reserve organs, the rhizophores. This work aimed at establishing a protocol for in vitro culture of V. herbacea, using seeds (achenes) and leaf discs as explants. Following germination and seedling growth, stem nodes from 6-month-old in vitro germinated plants were isolated and incubated on culture medium free of growth regulators for plant propagation and rhizophore formation. Fructan content and composition were evaluated in leaves, stems, roots and rhizophores from plants grown in vitro and compared with those of greenhouse-grown plants, in order to evaluate inulin production in vitro. Fructan contents of aerial organs and roots from in vitro plants were higher, compared with greenhouse plants, while in rhizophores, the opposite was observed. High performance anion exchange chromatography/pulsed amperometric detection profiles revealed the presence of the inulin homologous series in the aerial organs exclusively for in vitro plants, while in roots and rhizophores, this series was detected in plants grown in both conditions. These results indicate a modification in the source/sink ratio, leading to changes in the distribution of carbohydrates in in vitro plants. The leaf disc cultures on medium supplemented with indole-3-butyric acid induced the formation of roots (0.24, 0.49 µM) and friable callus (2.46 µM), while 6-benzylaminopurine (from 1.1 through 4.43 µM) induced compact callus. However, no shoot formation was observed. The use of seeds allowed the establishment of a protocol for in vitro culture and provides a model system for a better understanding of fructan metabolism in V. herbacea.     

Isolation and characterization of a new fimbrial antigen (CS1541) from a porcine enteroxigenic Escherichia coli O8: KX105 strain

Abstract A fimbrial antigen (CS1541) was extracted and purified from the porcine enterotoxigenic Escherichia coli strain 1541P (O8:K-:H9). CS1541 fimbriae appeared as long thin filaments 3–5 mm in diameter. CS1541 antigen consisted of two peptide bands of about 18 and 19 kDa as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. It was expressed both at 37°C and 15°C and did not demonstrate hemagglutinating properties. It was antigenically distinct from the fimbrial antigens K88, K99, F41, FY(Att25), F165, type 1, CFA-I, and CFA-II complex but demonstrated serological cross-reactions with the 987P fimbrial antigen.     

Isolation, culture and characterization of a new strain of Euglena gracilis

A new strain of Euglena gracilis Klebs has been isolated from a highly polluted river; it was named MAT. Strain growth in different culture media was evaluated under heterotrophic and autotrophic conditions. Total lipid, sugar, protein and chlorophyll a production were studied. Results obtained for MAT were compared with data obtained for a UTEX Culture Collection strain. Likewise, cells from both strains were bleached using streptomycin, and grown in the same media used for green samples. Both MAT and UTEX showed clear differences in their biochemical composition and growth rate depending on the media used. They also exhibited different growth patterns. E. gracilis medium proved to be the best culture environment for both strains either in autotrophic or heterotrophic conditions. Results show that basal contents of lipids, sugars, proteins and chlorophyll a vary depending on the strain, and thus values obtained for one strain do not apply to another. Moreover, strain origin may have an influence on the mechanisms of adaptation or defense developed by each sample.     

Establishment and characterization of Stevia rebaudiana (Bertoni) cell suspension culture: an in vitro approach for production of stevioside

A protocol has been standardized for establishment and characterization of cell suspension cultures of Stevia rebaudiana in shake flasks, as a strategy to obtain an in vitro stevioside producing cell line. The effect of growth regulators, inoculum density and various concentrations of macro salts have been analyzed, to optimize the biomass growth. Dynamics of stevioside production has been investigated with culture growth in liquid suspensions. The callus used for this purpose was obtained from leaves of 15-day-old in vitro propagated plantlets, on MS medium fortified with benzyl aminopurine (8.9 μM) and naphthalene acetic acid (10.7 μM). The optimal conditions for biomass growth in suspension cultures were found to be 10 g l^?1 of inoculum density on fresh weight basis in full strength MS liquid basal medium of initial pH 5.8, augmented with 2,4-dichlorophenoxy acetic acid (0.27 μM), benzyl aminopurine (0.27 μM) and ascorbic acid (0.06 μM), 1.0× NH₄NO₃ (24.7 mM), 3.0× KNO₃ (56.4 mM), 3.0× MgSO₄ (4.5 mM) and 3.0× KH₂PO₄ (3.75 mM), in 150 ml Erlenmeyer flask with 50 ml media and incubated in dark at 110 rpm. The growth kinetics of the cell suspension culture has shown a maximum specific cell growth rate of 3.26 day^?1, doubling time of 26.35 h and cell viability of 75 %, respectively. Stevioside content in cell suspension was high during exponential growth phase and decreased subsequently at the stationary phase. The results of present study are useful to scale-up process and augment the S. rebaudiana biological research.     

Isolation and characterisation of a strain of Saccharomyces cerevisiae deficient in in vitro RNA polymerase B(II) activity.

Summary Two hundred strains of Saccharomyces cerevisiae temperature sensitive for RNA synthesis were selected and screened in crude extracts for DNA-dependent RNA polymerase activities. One strain was isolated which had only residual in vitro RNA polymerase B activity. In normal growth conditions total RNA, poly A⁺ RNA and protein synthesis were indistinguishable from those of the wild type strain at 23°C and after shift to 37°C. A temperature sensitive phenotype was detected only when rpoB containing strains were grown in adverse conditions. The mutant character showed mendelian segregation and was coexpressed with the wild type character in heterozygous diploids. Residual enzyme activity was characterised in crude extracts using synthetic polymers and natural templates in different ionic conditions.     

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.     

凤梨科植物的离体培养（综述）

本文主要从快速繁殖,育种探索和种质保存三个方面介绍凤梨离体培养研究的进展,并简要介绍其他凤梨科植物离体培养的研究概况,在此基础上对一些问题进行探讨并提出展望。     

具有高木二糖形成活力的木聚糖酶生产菌株的筛选与产酶培养基的优化

从土样中筛选出一株产木聚糖酶的青霉,该青霉所产木降糖酶具有很高的木二糖形成活力,经鉴定为顶青霉,其木聚糖酶的合成与分泌受木聚糖等木糖苷类物质的诱导,麸皮对其木聚糖酶的合成也有促进作用,优化产酶液体培养主要成分的配比为：麸皮：玉米芯木聚糖：玉米芯粉;蛋白胨（或尿素）＝1：1：1：0．6（0．4）,摇瓶96h达到最大酶活,最高木聚糖酶活达到289．3U／ml,该菌所产木聚糖酶的最适作用条件为45－50度,PH4．4,在PH4．4－8．0范围内稳定。     

Isolation and purification of Rh(E) antigen

The Rh(E) antigen of human red blood cell membranes has been isolated. The method of preparation was as follows: Red cell membranes were solubilized using ethylenediaminetetraacetic acid followed by NaCl. Membrane ultrafilters were used to separate solubilized membrane components; the molecular weight class between 50 000 and 100 000 had Rh(E) activity. The Rh(E) antigen was eluted as a single component by isoelectric focusing using a gradient, pH 6–pH 8. The Rh(E) antigen inhibits the agglutination of antibody coated Rh(E) positive cells and gives a high titer of antibody in guinea pig.