Expression of C-terminal truncated and full-length Babesia bigemina rhoptry-associated protein 1 and their potential use in enzyme-linked immunosorbent assay

Recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of Babesia bigemina infection by using a full-length B. bigemina rhoptry-associated protein 1 (rRAP-1) and the truncated C-terminal RAP-1 (rRAP-1/CT). While the rRAP-1 showed cross reactivity between B. bigemina- and Babesia bovis-infected bovine sera, the rRAP-1/CT was highly specific to B. bigemina-infected bovine sera and proved useful in the detection of sequential sera collected from an experimentally infected cow during the acute and latent infection. The high yield of soluble rRAP-1/CT and its diagnostic specificity demonstrate its potential in the diagnosis of B. bigemina infection. Its usefulness for epidemiological investigation is currently being evaluated.     

Amiprophosmethyl-induced efficient in vitro production of polyploids in raphanobrassica with the aid of aminoethoxyvinylglycine (AVG) in the culture medium

Optimum conditions for obtaining tetraploid were investigated in raphanobrassica, the intergeneric hybrid between radish (Raphanus sativus) and kale (Brassica oleracea var. acephala) by treating in vitro plants with an anti-mitotic agent, amiprophosmethyl (APM). Initially, no tetraploids but hexaploids and octaploids were induced by the treatments. Although the leaves of these polyploids of raphanobrassica showed chlorosis during subcultures in in vitro conditions, the chlorosis could be successfully prevented by the ethylene inhibitors, both AVG and AgNO₃. Based on this result, AVG was added into medium used for the culture after the chromosome doubling treatment, which subsequently resulted in increased survival rates of the treated plant materials as well as increased production rates of polyploids including tetraploid. These polyploid plants showed obviously different characters from the original diploid plant. The tetraploid plant had bigger sizes in shoot, flower and leaf, and more number of leaves than the diploid. On the other hand, the hexaploid and octaploid plants had smaller sizes in shoots and leaves, and less number of leaves than the diploid. Concentration of glucosinolates, functional substances of Brassicaceae crops, did not significantly differ between diploid and tetraploid of raphanobrassica, but reduced in hexaploid and octaploid.     

Host status of 10 fungal isolates for two nematode species, Filenchus misellus and Aphelenchus avenae

The classification of nematodes in the family Tylenchidae into plant parasites, plant associates or fungal-feeders for community analyses, have been much discussed by nematode ecologists. For an appropriate classification, fungal-feeding habits in the family need to be studied. To evaluate the host status of 10 fungal isolates for Filenchus misellus (Tylenchidae) and Aphelenchus avenae (Aphelenchida, Aphelenchidae), population growth rates, body length and width and sex ratios of the nematodes were measured after 40-day culture on fungal colonies at 25 °C. For F. misellus, the fungi determined as good hosts were two Basidiomycota fungi (Agaricus bisporus, Coprinus cinereus), three Ascomycota fungi (Chaetomium cochlioides, Chaetomium funicola, Chaetomium globosum) and a plant-pathogenic fungus (Rhizoctonia solani) on the basis of nematode population growth rate and female body length. Interestingly Pleurotus ostreatus, known as a predaceous fungus for the other nematodes, was also a good host for F. misellus. While, for A. avenae, good hosts were four plant-pathogenic fungi (Fusarium oxysporum f. sp. conglutinans, F. oxysporum f. sp. cucumerinum, Pythium ultimum, R. solani) and A. bisporus. A. avenae was trapped and preyed upon by Pleurotus hyphae. In F. misellus, males were 7-21% of adults, but the ratio did not correlate significantly with the population growth rate. In A. avenae, no male occurred. Differences in habitat preference between Filenchus and Aphelenchus were explained on the basis of the host status and habitat preferences of the tested fungi.     

Development of a single-round and multiplex PCR method for the simultaneous detection of Babesia caballi and Babesia equi in horse blood

With the aim of developing more simple diagnostic alternatives, a differential single-round and multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of Babesia caballi and Babesia equi, by targeting 18S ribosomal RNA genes. The multiplex PCR amplified DNA fragments of 540 and 392 bp from B. caballi and B. equi, respectively, in one reaction. The PCR method evaluated on 39 blood samples collected from domestic horses in Mongolia yielded similar results to those obtained from confirmative PCR methods that had been established earlier. Thus, the single-round and multiplex PCR method offers a simple tool for the differential diagnosis of B. caballi and B. equi infections in routine diagnostic laboratory settings as well as in epidemiological studies.     

Utilization of genetically modified soybean meal in Nile tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis> diets

The utilization of genetically modified soybean meal (GM SBM) was compared with that of non-GM SBM in Nile tilapia. Four experimental diets were formulated to include either non-GM or GM SBM at 34 or 48%, respectively. These diets were fed to juvenile Nile tilapia (49.5 g average weight) for 12 weeks. The uptake of the cauliflower mosaic virus 35S promoter fragment of the GM SBM in fish muscle was examined at 8th and 12th week. After 12th week, fish were fed the non-GM SBM diets to determine the residual span of the incorporated promoter fragment. There was no significant difference in specific growth rate or feed efficiency between GM and non-GM groups at the same inclusion level. A small number of muscles from fish receiving both levels of GM SBM diet were positive for the promoter fragment. Additionally, the promoter fragment was not detected by the second day after changing to the non-GM SBM diets. These results indicate that the utilization of GM SBM was similar to that of non-GM SBM and the promoter fragment was rarely found in fish muscles, suggesting that suitability and safety of GM SBM in Nile tilapia diet were similar to those of non-GM SBM.     

Molecular epidemiological survey of Theileria orientalis in Thua Thien Hue Province, Vietnam

Theileria orientalis is a benign bovine protozoan parasite that occasionally causes serious economic loss in the livestock industry. We report the findings of a molecular epidemiological survey of T. orientalis in 94 Vietnamese yellow cattle, 43 water buffaloes, 21 sheep, 21 goats and 85 blood-sucking ticks of cattle in the Thua Thien Hue province of Vietnam. The major piroplasm surface protein (MPSP) gene of T. orientalis was detected using polymerase chain reaction from 13 cattle (13.8%), 11 water buffaloes (25.6%), 1 sheep (4.8%) and 9 ticks (10.6%). Phylogenetic analysis using MPSP gene sequences showed the presence of seven genotypes, four previously categorized genotypes (Types 1, 3, 5 and 7) and three new genotypes (Types N-1, N-2 and N-3).     

Serological survey of Babesia bovis and Babesia bigemina in cattle in South Africa

A total of 719 serum samples collected from clinically healthy cattle from eight provinces located in different districts of South Africa were examined by the indirect enzyme-linked immunosorbent assay (ELISA) and the standard indirect fluorescent antibody test (IFAT) to determine the serological prevalence of Babesia bovis and Babesia bigemina. The results showed that 35.3% and 39.7% of cattle were positive for B. bovis and 30% and 36.5% were positive for B. bigemina antibodies on ELISA and IFAT, respectively. Mixed infections were detected in 18.2% and 26.3% of the samples using ELISA and IFAT, respectively. Consequently, the ELISAs with recombinant B. bovis spherical body protein-4 (BbSBP-4) and B. bigemina C-terminal rhoptry-associated protein-1 (BbigRAP-1/CT) were proven to be highly reliable in the serological diagnoses of bovine babesiosis in South African cattle, as evidenced by the significant concordance rates when the results were compared to those of IFAT. Moreover, the serological prevalence was significantly different among the tested provinces, in which the ranges exhibited between 15% and 73% for B. bovis infection and between 13% and 54% for B. bigemina infection. High sero-positive rates were present in Mpumalanga and KwaZulu-Natal provinces, while the lowest rate was in the North West province. Our data provide important information regarding the current seroprevalence of bovine babesiosis in South Africa, which might be beneficial in developing rational strategies for disease control and management.     

Glycosylation of the envelope protein of West Nile Virus affects its replication in chicks

Birds are important for the transmission of West Nile virus (WNV) in nature, but the significance of the potential N-linked glycosylation at position 154 in the WNV envelope (E) protein with regard to viral replication in young chickens has not been assessed. In this study, the effect of glycosylation of the WNV E protein on viral pathogenicity in birds was investigated using young domestic chicks. A higher viral load was detected in the blood and the peripheral organs, particularly the hearts, of 2-day-old chicks inoculated with a glycosylated WNV variant compared to those inoculated with the nonglycosylated variant. There was no significant difference in the neutralizing antibody titers and cytokine expression profiles in chickens inoculated with the glycosylated and the nonglycosylated WNV variants. In contrast, no virus w as detected in the blood and the tissues of 3-wk-old chicks, although the host immune response was induced to similar levels as in the 2-day-old chicks. These data indicate the utility of young domestic chicks as an animal model of WNV infection; they also indicate that glycosylation of the E protein of WNV enhances multiplication in the blood and peripheral organs, which is associated with the strong pathogenicity of WNV in birds.     

Water-soluble organic materials in paddy soil ecosystem

Abstract

A column experiment was conducted to analyse the composition of organic materials in the leachate from the plow layer and their fate in the subsoil. Water-soluble organic materials in the leachate were fractionated by insoluble polyvinylpyrrolidone (PVP) and ion exchange resins. The content of total organic carbon (TOC) in the leachate increased by the addition of rice straw (RS) to the plow layer soil sample. The leachate contained a constant amount of PVP-adsorbed Fraction, while that of the PVP-non-adsorbed Fraction changed during the 45 day incubation period. In the fractionation using ion exchange resins, the fraction adsorbed onto the anion exchange resin was the major one.

By the connection of a subsoil column to the plow layer soil column with RS, the TOC content in the leachate decreased by percolation into the subsoil sample. In the Anjo soil sample (Yellow Soil), the decrease occurred throughout the incubation period, and about 90% of the PVP-adsorbed Fraction in the leachate decreased by percolation into the subsoil sample. In the Fukushima soil sample (Gray Lowland Soil), the TOC content decreased in the early and middle periods of incubation, while in the late period the decrease was negligible. This decrease of the TOC content by percolation into the subsoil sample was mainly due to retention in the subsoil sample of the Anjo soil, while in the Fukushima soil sample it was due to decomposition and retention. It was considered that easily decomposable organic materials like organic acids were decomposed in the early to middle periods of incubation, while in the late period the contents of such substances in the leachate from the plow layer soil sample with RS were small and the decrease of TOC was negligible.     

cDNA microarray analysis of interleukin-1β-induced Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> kidney cells

ABSTRACT:   Interleukin-1β (IL-1β) cDNA of Japanese flounder was found to consist of 1329 bp, encoded 247 amino acid residues. Among the fish IL-1β in the databases, the one with the highest identity of Japanese flounder IL-1β was that of seabass (62% identity). The expression of IL-1β was induced by treatment with concanavalin A (ConA)/phorbol myristate acetate (PMA) and lipopolysaccharide. The copy number of IL-1β mRNA was increased 30-fold after stimulation with ConA/PMA. Of 871 cDNA on a microarray, 93 genes (10.7%) were up-regulated or down-regulated by IL-1β at 1, 3 and 7 days post-injection. The induced gene expression was highest on day 1 followed by day 3 and day 7. A total of 7% of known and 3.7% of unknown genes of the 871 tested genes were differentially expressed. Of the genes tested, 7.4% were up-regulated and 3.3% were down-regulated. Cytokine genes such as tumor necrosis factor, granulocyte colony stimulating factor and chemokine receptor A were induced in response to IL-1β. Cell surface antigens such as IgM, MHC class I and CD20 receptor were up-regulated. Signal transduction genes such as Toll-like receptor 1 and SH3P2 were also up-regulated. The glucocorticoid receptor and cAMP early repressor were down-regulated in our microarray analysis.