A Set of Lotus japonicus Gifu x Lotus burttii Recombinant Inbred Lines Facilitates Map-based Cloning and QTL Mapping

Model legumes such as Lotus japonicus have contributed significantly to the understanding of symbiotic nitrogen fixation. This insight is mainly a result of forward genetic screens followed by map-based cloning to identify causal alleles. The L. japonicus ecotype 'Gifu' was used as a common parent for inter-accession crosses to produce F2 mapping populations either with other L. japonicus ecotypes, MG-20 and Funakura, or with the related species L. filicaulis. These populations have all been used for genetic studies but segregation distortion, suppression of recombination, low polymorphism levels, and poor viability have also been observed. More recently, the diploid species L. burttii has been identified as a fertile crossing partner of L. japonicus. To assess its qualities in genetic linkage analysis and to enable quantitative trait locus (QTL) mapping for a wider range of traits in Lotus species, we have generated and genotyped a set of 163 Gifu × L. burttii recombinant inbred lines (RILs). By direct comparisons of RIL and F2 population data, we show that L. burttii is a valid alternative to MG-20 as a Gifu mapping partner. In addition, we demonstrate the utility of the Gifu × L. burttii RILs in QTL mapping by identifying an Nfr1-linked QTL for Sinorhizobium fredii nodulation.     

Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing

In a previous report (Luyo-Acero et al., 2004), we demonstrated that cytochrome b (Cyt b) gene analysis is an effective method for classifying several isolates of the genus Leishmania; hence, we have further applied this method to other Leishmania species in an effort to enhance the accuracy of the procedure and to construct a new phylogenic tree. In this study, a total of 30 Leishmania and Endotrypanum WHO reference strains, clinical isolates from our patients assigned to 28 strains (human and non-human pathogenic species) and two species of the genus Endotrypanum were analyzed. The Cyt b gene in each sample was amplified by PCR, and was then sequenced by several primers, as reported previously. The phylogenic tree was constructed based on the results obtained by the computer software MEGA v3.1 and PAUP* v4.0 Beta. The present phylogenic tree was almost identical to the traditional method of classification proposed by Lainson and Shaw (1987). However, it produces the following suggestions: (1) exclusion of L. (Leishmania) major from the L. (L.) tropica complex; (2) placement of L.tarentolae in the genus Sauroleishmania; (3) L. (L.) hertigi complex and L. (V.) equatorensis close to the genus Endotrypanum; (4) L. (L.) enrietti, defined as L. (L.) mexicana complex, placed in another position; and (5) L. (L.) turanica and L. (L.) arabica are located in an area far from human pathogenic Leishmania strains. Cyt b gene analysis is thus applicable to the analyzing phylogeny of the genus Leishmania and may be useful for separating non-human pathogenic species from human pathogenic species.     

Genetic variation and relationships in Laetiporus sulphureus s. lat., as determined by ITS rDNA sequences and in vitro growth rate

The aim of this study was to characterise the genetic variation and molecular relationships of the brown rot polypore, Laetiporus sulphureus s. lat., from Europe, South America, Africa, and Asia, using ITS sequences of the nu-rDNA and by comparing the growth rate in vitro. In a NJ analysis of the sequences of 130 individuals of L. sulphureus s. lat., eight distinct clusters emerged, supported by BS values of 70-100 %. Within each cluster, the ITS rDNA sequence variation was below 3 %. The sequences were also analysed together with Laetiporus sequences available from GenBank. Results demonstrated the possible presence of L. huroniensis in Europe (invalidly named L. montanus) and L. gilbertsonii in South America, and provided more information on the Pan-American and European distribution of one of the clades, currently known in North America as L. sulphureus. L. conifericola formed a separate distinct clade. Moreover, the analysis revealed two unknown Laetiporus taxa in Korea, one in South Africa, and one in Europe. As L. sulphureus is described from Europe (France), and we show that more than one taxon exist here, it is presently not possible to define L. sulphureus s. str. Certain biological differences between some of the clades (in vitro growth rates, chemical composition, and pigmentation) were demonstrated and discussed.     

NMR spectroscopic analysis of exopolysaccharides produced by Leuconostoc citreum and Weissella confusa

Dextrans are the main exopolysaccharides produced by Leuconostoc species. Other dextran-producing lactic acid bacteria include Streptococci, Lactobacilli, and Weissella species. Commercial production and structural analysis has focused mainly on dextrans from Leuconostoc species, particularly on Leuconostoc mesenteroides strains. In this study, we used NMR spectroscopy techniques to analyze the structures of dextrans produced by Leuconostoc citreum E497 and Weissella confusa E392. The dextrans were compared to that of L. mesenteroides B512F produced under the same conditions. Generally, W. confusa E392 showed better growth and produced more EPS than did L. citreum E497 and L. mesenteroides B512F. Both L. citreum E497 and W. confusa E392 produced a class 1 dextran. Dextran from L. citreum E497 contained about 11% alpha-(1-->2) and about 3.5% alpha-(1-->3)-linked branches whereas dextran from W. confusa E392 was linear with only a few (2.7%) alpha-(1-->3)-linked branches. Dextran from W. confusa E392 was found to be more linear than that of L. mesenteroides B512F, which, according to the present study, contained about 4.1% alpha-(1-->3)-linked branches. Functionality, whether physiological or technological, depends on the structure of the polysaccharide. Dextran from L. citreum E497 may be useful as a source of prebiotic gluco-oligosaccharides with alpha-(1-->2)-linked branches, whereas W. confusa E392 could be a suitable alternative to widely used L. mesenteroides B512F in the production of linear dextran.     

Protein disulphide isomerase of Ostertagia ostertagi: an excretory-secretory product of L4 and adult worms?

A pepstatin A-agarose column was used in an attempt to purify a previously described antibody-degrading aspartyl proteinase from excretory-secretory material from the L4 and the adult stages of the bovine abomasal nematode Ostertagia ostertagi. However, no aspartyl proteinase activity was detected in the eluted fractions (L4Pepst and AdPepst). Screening of cDNA libraries with polyclonal antibodies raised against L4Pepst and AdPepst showed that a protein disulphide isomerase (Ost-PDI2) was present in both antigen fractions. This multifunctional enzyme was detected in extracts of L3, L4 and adult parasites and, interestingly, also in excretory-secretory material of L4 and adult O. ostertagi. By immunohistochemistry, the Ost-PDI2 enzyme was localised in some parts of the hypodermis of L4 and adult worms and in the intestinal cells of all three parasitic life stages. Two-dimensional Western blot analysis indicated that Ost-PDI2 is recognised by calves during a natural O. ostertagi infection, which suggests that Ost-PDI2 could be used for immunological control of ostertagiosis.     

Miltefosine-induced apoptotic cell death on Leishmania major and L. tropica strains

The aim of this study was to assess the cytotoxic effects of various concentrations of miltefosine on Leishmania major (MRHO/IR/75/ER) and L. tropica (MHOM/IR/02/Mash10) promastigotes and to observe the programmed cell death features. The colorimetric MTT assay was used to find L. major and L. tropica viability and the obtained results were expressed as 50% inhibitory concentration (IC50). Also, 50% effective doses (ED50) for L. major and L. tropica amastigotes were also determined. Annexin-V FLUOS staining was performed to study the cell death properties of miltefosine using FACS analysis. Qualitative analysis of the total genomic DNA fragmentation was performed by agarose gel electrophoresis. Furthermore, to observe changes in cell morphology, promastigotes were examined using light microscopy. In both strains of L. major and L. tropica, miltefosine induced dose-dependent death with features of apoptosis, including cell shrinkage, DNA laddering, and externalization of phosphatidylserine. The IC50 was achieved at 22 μM and 11 μM for L. major and L. tropica after 48 hr of incubation, respectively. ED50 of L. major and L. tropica amastigotes were 5.7 μM and 4.2 μM, respectively. Our results indicate that miltefosine induces apoptosis of the causative agent of cutaneous leishmaniasis in a dose-dependent manner. Interestingly, L. major did not display any apoptotic changes when it was exposed to miltefosine in concentrations sufficient to kill L. tropica.     

水产用聚维酮碘对异育银鲫养殖的安全性评价

评价水产用聚维酮碘对异育银鲫(Carassius auratus gibelio)养殖的安全性,为其在异育银鲫养殖中的安全应用提供了重要的科学依据,本研究参照国家标准及相关法规,在观察了聚维酮碘对小球藻(Chlorella sp.)生长抑制作用、对水产益生菌抑菌效果以及对大型蚤(Daphnia magna straus)、斑马鱼(Brachydanio rerio)和异育银鲫的急性毒性的基础上,分析其对异育银鲫及其养殖水体主要有害理化因子的影响.实验结果表明,聚维酮碘在终浓度为6.00 ～ 14.00 mg/L时对小球藻生长具有促进作用,对小球藻的半数抑制浓度大于14.00 mg/L,对水产益生菌的最小抑菌浓度为128～512 mg/L,对大型蚤、斑马鱼的半数致死浓度分别为13.44 mg/L、17.63 mg/L.此外,聚维酮碘对异育银鲫的半数致死浓度为74.77 mg/L,而且在养殖水体中加入聚维酮碘至终浓度为0.20 ～ 1.40 mg/L后14 d内,随着聚维酮碘浓度的增加,各浓度组异育银鲫养殖水体的氨氮含量、亚硝酸盐含量均缓慢下降.本研究证实聚维酮碘低毒,但考虑到其可能对异育银鲫养殖水体中大型蚤等浮游动物存在潜在影响,建议其在异育银鲫养殖中的安全应用浓度应不高于1.34 mg/L,在该安全应用浓度内不会引起养殖水中氨氮、亚硝酸盐等有害因子含量的增加.     

A role for the C. elegans L1CAM homologue lad-1/sax-7 in maintaining tissue attachment

The L1 family of cell adhesion molecules (L1CAMs) is important for neural development. Mutations in one of the human L1CAM genes, L1, can result in several neurological syndromes, the symptoms of which are variably penetrant. The physiological cause of these symptoms, collectively termed CRASH, is not clear. Caenorhabditis elegans animals genetically null for the L1CAM homologue LAD-1, exhibit variably penetrant pleiotropic phenotypes that are similar to the CRASH symptoms; thus the C. elegans lad-1 mutant provides an excellent model system to study how disruption of L1 leads to these abnormalities. These phenotypes include uncoordinated movements, variable embryonic lethality, and abnormal neuronal distribution and axon trajectories. Our analysis revealed that many of these phenotypes are likely a result of tissue detachment.     

Paramphistomum daubneyi: characteristics of infection in three lymnaeid species

Experimental infections of two South American lymnaeids (Lymnaea neotropica and L. viatrix var. ventricosa) with Paramphistomum daubneyi were carried out to determine if these snail species could sustain larval development of this digenean and, if so, to specify their potential for cercarial production. A French population of Galba truncatula infected and raised according to the same protocol served as controls. In both experiments, prevalence of P. daubneyi infections in snails did not significantly differ from each other. In snail groups evaluated for cercarial shedding (first experiment), a significantly lower number of shed cercariae was noted for L. neotropica, while those from G. truncatula and L. v. ventricosa did not differ significantly from each other. Dissection of infected snails at day 65 post-exposure at 20 °C (second experiment) found significantly lower burdens of P. daubneyi rediae and cercariae in the bodies of L. neotropica than in those of G. truncatula and L. v. ventricosa. Compared to total cercarial production observed in dissected snails, the percentage of cercariae which exited from snails was 75.6% for G. truncatula, 21.6% for L. neotropica, and 91.4% for L. v. ventricosa. This last species seems to be a good candidate for metacercarial production of P. daubneyi.     

Effects of temperature, pH and salinity on the infection of Leptolegnia chapmanii Seymour (Peronosporomycetes) in mosquito larvae

The effects of temperature, pH, and NaCl concentrations on the infectivity of zoospores of Leptolegnia chapmanii (Argentine isolate) were determined for Aedes aegypti and Culex pipiens under laboratory conditions. Zoospores of L. chapmanii were infectious at temperatures between 10 and 35 degrees C but not at 5 or 40 degrees C. At the permissive temperatures, mortality rates in young instars were much higher than in older instars and larvae of Ae. aegypti were more susceptible to L. chapmanii than larvae of Cx. pipiens. At 25 degrees C, Ae. aegypti larvae challenged with L. chapmanii zoospores resulted in 100% infection at pH levels ranging from 4 to 10. Larvae of Cx. pipiens exposed to similar pH and zoospore concentrations resulted in increasing mortality rates from 62% to 99% at pH 4 to 7, respectively, and then decreased to 71% at pH 10. Aedes aegypti larvae exposed to L. chapmanii zoospores in NaCl concentrations ranging from 0 to 7 parts per thousand (ppt) at 25 degrees C resulted in 100% mortality while mortality rates for Cx. pipiens decreases from 96% in distilled water to 31.5% in water with 6 ppt NaCl. Control Cx. pipiens larvae died when exposed at a NaCl concentration of 7 ppt. Vegetative growth of L. chapmanii was negatively affected by NaCl concentrations. These results have demonstrated that the Argentinean isolate of L. chapmanii tolerated a wide range of temperatures, pH, and salinity, suggesting that it has the potential to adapt to a wide variety of mosquito habitats.     

Effect of the exposure to Fasciola hepatica (Trematoda: Digenea) on life history traits of Lymnaea cousini and Lymnaea columella (Gastropoda: Lymnaeidae)

The snails Lymnaea columella and Lymnaea cousini have both been reported as intermediate hosts of Fasciola hepatica in Colombia. The effect of the exposure to the parasite on survival, fecundity and size of these snails was evaluated by means of experimental infections and the life history traits of control and exposed groups were compared. Infection rates were 82.2 and 34% for L. columella and L. cousini, respectively. A reduction in fitness was observed in both species when exposed to the parasite: fecundity alone was reduced in L. columella whereas in L. cousini there was also a decline in survival rate. Unlike other studies, increased size was not observed in either species. On the contrary, a reduction in growth rate was observed in L. columella.     

Development of species-specific PCR and PCR-restriction fragment length polymorphism assays for L.infantum/L.donovani discrimination

Discrimination of Leishmaniainfantum and L. donovani, the members of the L. (L.) donovani complex, is important for diagnosis and epidemiological studies of visceral leishmaniasis (VL). We have developed two molecular tools including a restriction fragment length polymorphisms of amplified DNA (PCR-RFLP) and a PCR that are capable to discriminate L. donovani from L. infantum. Typing of the complex was performed by a simple PCR of cysteineproteaseB (cpb) gene followed by digestion with DraIII. The enzyme cuts the 741-bp amplicon of L. donovani into 400 and 341 bp fragments whereas the 702 bp of L. infantum remains intact. The designed PCR species-specific primer pair is specific for L. donovani and is capable of amplifying a 317 bp of 3’ end of cpb gene of L. donovani whereas it does not generate an amplicon for L. infantum. The species-specific primers and the restriction enzyme were designed based on a 39 bp insertion/deletion (indel) in the middle of the cpb gene. Both assays could differentiate correctly the two species and are reliable and high-throughput alternatives for molecular diagnosis and epidemiological studies of VL in various foci.     

Efficient germ-line transformation of the economically important pest species Lucilia cuprina and Lucilia sericata (Diptera, Calliphoridae)

The green blowfly species Lucilia cuprina and Lucilia sericata are economically important pests for the sheep industries of Australia and New Zealand. L. cuprina has long been considered a good target for a genetic pest management program. In addition, L. sericata maggots are used in the cleaning of wounds and necrotic tissue of patients suffering from ulcers that are difficult to treat by other methods. Development of efficient transgenesis methods would greatly facilitate the development of strains ideal for genetic control programs or could potentially improve “maggot therapy”. We have previously reported the germ-line transformation of L. cuprina and the design of a “female killing system” that could potentially be applied to this species. However, the efficiency of transformation obtained was low and transformed lines were difficult to detect due to the low expression of the EGFP marker used. Here we describe an efficient and reliable method for germ-line transformation of L. cuprina using new piggyBac vector and helper plasmids containing the strong promoter from the L. cuprina hsp83 gene to drive expression of the transposase and fluorescent protein marker gene. We also report, for the first time, the germ-line transformation of L. sericata using the new piggyBac vector/helper combination.     

Nitrogen excretion by the sheep abomasal parasite Teladorsagia circumcincta

Excretion of nitrogenous substances by Teladorsagia circumcincta was investigated during incubation of L3 in phosphate buffer for up to 30 h and adult worms for 4-6 h. Ammonia was the main excretory product, with about 20% urea. For the first 4-6 h, ammonia excretion by L3 was temperature dependent, directly proportional to the number of larvae, but independent of the pH or strength of the phosphate buffer. Later, ammonia excretion slowed markedly in L3 and adults and reversed to net uptake in L3 by 30 h. An initial external ammonia concentration of 600 μM did not alter the pattern or magnitude of excretion. Re-uptake of ammonia did not occur at extremes of pH or low buffer strength and was slightly reduced at the highest external concentrations. Ammonium transporters and enzymes of glutamate metabolism, including glutamate dehydrogenase, glutamine synthetase and possibly glutamate synthase, are worthy of further investigation as anthelmintic targets.     

Leishmania species: Detection and identification by nested PCR assay from skin samples of rodent reservoirs

Many rodent species act as reservoir hosts of zoonotic cutaneous leishmaniasis in endemic areas. In the present study a simple and reliable assay based on nested PCR was developed for the detection and identification of Leishmania parasites from rodent skin samples. We designed Leishmania-specific primers that successfully amplified ITS regions of Leishmania major, Leishmania gerbilli and Leishmania turanica using nested PCR. Out of 95 field collected Rhombomys opimus, 21 were positive by microscopic examination and 48 by nested PCR. The percentage of gerbils infected with L. major, L. gerbilli and L. turanica was 3.2%, 1.1% and 27.4%, respectively. In 15.8% of the rodents, we found mixed natural infections by L. major and L. turanica, 1.1% by L. major and L. gerbilli, and 2.1% by the three species. We concluded that this method is simple and reliable for detecting and identifying Leishmania species circulating in rodent populations.     

Serosurveillance of Scrub Typhus in Small Mammals Collected from Military Training Sites near the DMZ, Northern Gyeonggi-do, Korea, and Analysis of the Relative Abundance of Chiggers from Mammals Examined

Comprehensive quarterly serosurveillance on scrub typhus in small mammals collected from military training sites located near the Demilitarized Zone (DMZ), northern Gyeonggi-do (Province), ROK was conducted to determine the potential rodent-borne and associated ectoparasite disease risks to military personnel. A total of 1,196 rodents and insectivores representing 8 species, Apodemus agrarius (87.3%, n = 1,044), Mus musculus (5.4%, n = 65), Crocidura lasiura (3.3%, n = 40), Microtus fortis (2.6%, n = 31), Micromys minutus (0.3%, n = 4), Tscherskia triton (0.3%, n = 4), Rattus norvegicus (0.3%, n = 4), and Myodes regulus (0.3%, n = 4) were assayed for the presence of antibodies to Orientia tsutsugamushi. O. tsutsugamushi antibodies were detected in 6 of 8 species and seroprevalence determined; A. agrarius (45.6%), M. musculus (23.1%), M. fortis (48.4%), M. minutus (50.0%), T. triton (50.0%), and R. norvegicus (25.0%). A total of 31,184 chigger mites collected from 508 rodents and insectivores were slide-mounted and 10 species belonging to 4 genera were identified. Leptotrombidium pallidum (53.4%) was the most frequently collected, followed by L. palpale (15.7%), Neotrombicula tamiyai (14.3%), L. orientale (10.7%), L. zetum (3.1%), Walchia fragilis (2.1%), and L. gemiticulum (0.8%), while the remaining 3 species, L. subintermedium, N. gardellai, and Euschoengastia koreaensis were rarely observed (prevalence < 10%). In contrast to previous surveys, higher chigger indices of the primary scrub typhus vectors, L. pallidum (165.4), L. orientale (45.0), and L. palpale (21.4), were observed during the spring season.     

Partial protective responses induced by a recombinant cysteine proteinase from Leishmania (Leishmania) amazonensis in a murine model of cutaneous leishmaniasis

A 500 bp fragment encoding an isoform of cysteine proteinase from Leishmania (Leishmania) amazonensis was subcloned and expressed in the pHis vector, resulting in a recombinant protein of 24 kDa, rLacys24. In Western blots of L. (L.) amazonensis extracts, antibodies directed to rLacys24 recognized a cysteine proteinase isoform of 30 kDa. Analysis by fluorescence-activated cell sorter showed a significantly higher expression of CD8⁺ lymphocytes in animals immunized with rLacys24 plus CFA, whereas a low expression of CD4⁺ lymphocytes was observed in these animals. The cytotoxicity of lymphocytes isolated from mice immunized with rLacys24 plus CFA on L. (L.) amazonensis-infected macrophages was significantly higher than that observed in the presence of lymphocytes from control animals. Immunization of BALB/c mice with rLacys24 plus CFA resulted in a low but significant decrease of foot lesions after challenge with L. (L.) amazonensis compared to those exhibited by control mice.