Survey of plasmids and resistance factors among veterinary isolates of Salmonella enteritidis in Malaysia

Thirty-five veterinary isolates of Salmonella enteritidis were characterized by their susceptibility to 10 antimicrobial agents and by their plasmid profiles on agarose gel electrophoresis. All were susceptible to carbenicillin, chloramphenicol and nalidixic acid but 89% were resistant to tetracycline. When examined, 91% of the isolates harboured plasmids, with sizes ranging from 9.8 to 60 MDa. However, it was only possible to associate the presence of plasmids with tetracycline resistance; plasmids occurring in 90% of the tetracycline-resistant isolates. In conjugation experiments, with Escherichia coli K12 Nal^r as recipient, the tetracycline resistance in three selected S. enteritidis isolates was observed to transfer at frequencies of 3.0×10^-3 to 1.0×10^-2/donor cell. The concomitant transfer of a 56-MDa or 60-MDa plasmid in these three S. enteritidis isolates was also detected.R. Son. A. Ansary and I. Salmah are with the Department of Genetics and Cellular Biology. University of Malaya, 59100 Kuala Lumpur, Malaysia     

Comparative microsatellite typing of new world leishmania infantum reveals low heterogeneity among populations and its recent old world origin

Leishmania infantum (syn. L. chagasi) is the causative agent of visceral leishmaniasis (VL) in the New World (NW) with endemic regions extending from southern USA to northern Argentina. The two hypotheses about the origin of VL in the NW suggest (1) recent importation of L. infantum from the Old World (OW), or (2) an indigenous origin and a distinct taxonomic rank for the NW parasite. Multilocus microsatellite typing was applied in a survey of 98 L. infantum isolates from different NW foci. The microsatellite profiles obtained were compared to those of 308 L. infantum and 20 L. donovani strains from OW countries previously assigned to well-defined populations. Two main populations were identified for both NW and OW L. infantum. Most of the NW strains belonged to population 1, which corresponded to the OW MON-1 population. However, the NW population was much more homogeneous. A second, more heterogeneous, population comprised most Caribbean strains and corresponded to the OW non-MON-1 population. All Brazilian L. infantum strains belonged to population 1, although they represented 61% of the sample and originated from 9 states. Population analysis including the OW L. infantum populations indicated that the NW strains were more similar to MON-1 and non-MON-1 sub-populations of L. infantum from southwest Europe, than to any other OW sub-population. Moreover, similarity between NW and Southwest European L. infantum was higher than between OW L. infantum from distinct parts of the Mediterranean region, Middle East and Central Asia. No correlation was found between NW L. infantum genotypes and clinical picture or host background. This study represents the first continent-wide analysis of NW L. infantum population structure. It confirmed that the agent of VL in the NW is L. infantum and that the parasite has been recently imported multiple times to the NW from southwest Europe.     

Internuclear transfer of genetic information in kar1-1/KAR1 heterokaryons in Saccharomyces cerevisiae.

Heterokaryons of Saccharomyces cerevisiae have been constructed utilizing the kar1-1 mutation, which prevents nuclear fusion during conjugation (J. Conde and G. Fink, Proc. Natl. Acad. Sci. U.S.A. 73:3651-3655, 1976). Each heterokaryon contained two haploid nuclei that were marked on several chromosomes. They segregated haploid progeny (cytoductants), most of which have the nuclear genotype of one or the other of the heterokaryon parents, but they occasionally segregated progeny having a recombinant genotype (exceptional cytoductants). Exceptional cytoductants receive the majority of their genome from one parent (the recipient) and a minority from the other (the donor). Transfer of two markers from the donor nucleus to the recipient is rarely coincident for markers located on different chromosomes but is nearly always coincident for those markers located on the same chromosome, suggesting that whole chromosomes are transferred from the donor nucleus to the recipient. In crosses of kar1-1 X KAR1 parents, either nucleus may act as a recipient or donor with equal probability. Recipient nuclei acquired 9 of the 10 chromosomes examined, with frequencies which were inversely correlated with the size of the chromosome. When a chromosome is acquired by the recipient nucleus, it either replaces its homolog or exists in a disomic condition. Haploid progeny emanating from kar1 X KAR1 crosses are frequently inviable. I tested whether this inviability might be the result of chromosome loss by donor nuclei. Viability of progeny from kar1 X KAR1 heterokaryons was improved when the parental nuclei were diploid to an extent consistent with the hypothesis, and diploid progeny which had become monosomic were recovered from these heterokaryons. The following sequence of events accounts for chromosome transfer in kar1 X KAR1 heterokaryons. After cell fusion, each nucleus in the heterokaryon has a probability of about 0.38 of losing one or more chromosomes. A nucleus sustaining such a loss can become a donor in a chromosome transfer event. If the other nucleus does not sustain a mortal chromosome loss, it can become a recipient in a transfer event. The chance of acquiring a chromosome lost by the donor is greater for smaller chromosomes than for larger ones and is about 0.05 for the average chromosome.     

Transferable tetracycline resistance in Clostridium perfringens strains of porcine origin

These studies represent the first systematic survey of the incidence of conjugative antibiotic resistance in Clostridium perfringens. Ninety-two antibiotic-resistant porcine strains were examined to see if they could donate their antibiotic-resistance determinants to sensitive recipient strains. Fifteen of the 89 tetracycline-resistant strains transferred their tetracycline resistance in mixed-plate mating experiments but no transfer of macrolide-lincosamide resistance was detected. The efficiencies of transfer of tetracycline resistance varied from 1.3 X 10(-3) to 1.9 X 10(-6) transconjugants per donor cell. Significantly higher transfer efficiencies were observed when both the donor and recipient strains were derivatives of strain CW 362. These values ranged from 3.7 X 10(-1) to 4.6 X 10(-2) transconjugants per donor cell. This high frequency transfer system should prove invaluable for further genetic studies on this microorganism.     

Identification of some Bacteria from Paddy Antagonistic to Several Rice Fungal Pathogens

The effect of 23 bacterial strains from ricefields in the tropics on rice seed germination and on radicle and hypocotyl development of four rice cultivars was determined. There was a varietal difference in response to seed bacterization with the different bacterial strains. Germination of cv. IR58 increased from 78 to 93 %, that of cv. IR64, from 89 to 97 %. Less effects on germination of cvs IR42 and IR36 were observed. All strains inhibited the mycelial growth of Rhizoctonia solani in vitro. The three strains, identified as Bacillus subtilis, inhibited the mycelial growth of eight fungal pathogens whereas the other strains were pathogen-specific. Seed bacterization with these bacterial strains provided a sheath blight protection of 4. 5 to 73 % in the glasshouse trial. These 23 bacterial strains were identified by phenotypic tests using the API systems, morphological and biochemical features, and by comparison of electrophoretic patterns after sodium dodecyl sulphate polyacrylamide gel electrophoresis. Bacterial strains were identified (number of strains in brackets) as: Bacillus subtilis (3), Bacillus laterosporus (1), Bacillus pumilus (1), Pseudomonas aeruginosa (7), Pseudomonas belonging to section 1 (5), Erwina herbicola-like (1), and Serratia marcescens (1). The features of the other four strains were similar to Serratia except for the DNAase and lipase activities.     

Homology in capsular transformation reactions in Pneumococcus

Summary Experiments were carried out to determine the relative effect of homology inside or outside of the capsular genomes of donor and recipient strains of pneumococci on the frequency of transfer of capsular markers. In one series of experiments, 3 recipient strains were transformed to CapIII⁺ by DNA from 2 donor strains. Recipient strains (III)capIII D6 ¹, (II)capIII D15 P1 ¹, and (II)capII-1 ¹ were each transformed to CapIII⁺ at different absolute frequencies dependent upon the amount of genetic information that the strain had to acquire. The chromosomal background of the donor strain carrying the CapIII capsular genome had no influence on the results, however, for each strain was transformed at the same frequency by DNA from donor strain (II)CapIII⁺ or donor strain (III)CapIII⁺. In a second series of experiments, 2 (I)CapIII-strains, a (II)CapIII-strain and a (III)CapIII-strain were transformed to heterologous type I and binary type I-III with DNA from donor strains (I)CapI⁺, (II)CapI⁺, and (III)CapI⁺. Again, the chromosomal background of the donor strain was unimportant to the results. The origin of the recipient strain, however, markedly influenced the frequency of transformation. (I)CapIII-strains were transformed to CapI⁺ at about 10 times the frequency and to CapI-III at from 18–6000 times the frequency of the other CapIII-strains. Consideration of the results leads to the conclusion that transformation of CapIII-strains to CapI⁺ and transformation of CapI-strains to CapIII⁺ are not reciprocal reactions; CapI-strains lose less information in transformation to CapIII⁺ than CapIII-strains gain in transformation to CapI⁺. In (I)CapIII-recipient strains, the residual information from the CapI capsular genome is responsible for the higher frequency of transformation to both CapI⁺ and to CapI-III. It is suggested that addition of exogenous linear DNA to a recipient chromosome to give rise to binary strains occurs when sequence homology with the recipient is limited to one end of a piece of transforming DNA. Models to explain the results (Figs. 1 through 3) are consistent with the experimental findings and are amenable to further testing.     

Impact of defoliation by the biocontrol agentZygogramma bicolorataon the weed Partheniumhysterophorus in Australia

The leaf-feeding beetle Zygogrammabicolorata Pallister was introduced from Mexico intoAustralia in 1980 as a biocontrol agent for the weedParthenium hysterophorus L. (Asteraceae). Z. bicolorata became abundant in 1990, and since 1992there has been regular outbreaks resulting in thedefoliation of the weed in central Queensland. In thisstudy we evaluated the impact of defoliation by Z. bicolorata on P. hysterophorus from 1996 to1998. Z. bicolorata caused 91–100% defoliationresulting in reductions in weed density by 32–93%,plant height by 18–65%, plant biomass by 55–89%,flower production by 75–100%, soil seed-bank by13–86% and seedling emergence in the following seasonby 73–90%. At sites with continued outbreaks ofZ. bicolorata, it is expected that the existing soilseed-bank will be minimised, resulting in reduceddensity of parthenium in 6 to 7 years.     

Genotyping of Trichophyton rubrum by analysis of ribosomal-DNA intergenic spacer regions

An attempt was made to explore the genotyping of Trichophyton rubrum (T. rubrum) and the relationship between genotype and geographical origin using ribosomal restriction endonuclease polymorphic analysis. The total DNA was extracted by cetyltrimethyl ammonium bromide (CTAB). The probe was amplified from part of the 18S, ITSI, 5.8S, and ITSII region of T. rubrum standard strain with the universal fungal primers NS5 [5′-AACTT AAAGG AATTG ACGGA AG-3′] and ITS4 [5′-TCCTC CGCTT ATTGA TATGC-3′]. The genomic DNA of 49 clinical T. rubrum isolates digested by EcoR1 were hybridized with this probe, and the hybridization patterns were used as the basis of genotyping. Of the data from 49 strains of T. rubrum studied (21 from Nanjing, 26 from Dalian, and two from Beijing), 20 individual patterns (DNA Type A–T) were identified, among which Type A–C accounted for 48.98% of all the strains. The DNA patterns of Nanjing strains were represented by three bands, those of Dalian strains were represented by four bands. The DNA typing of T. rubrum by Southern blotting was highly sensitive and highly distinguishable. The DNA patterns of Nanjing strains were obviously different from those of Dalian strains.     

Nuclear selection in monokaryotization of dikaryotic mycelia ofPholiota nameko as described by leading and following nuclei

To examine monokaryotization of dikaryotic mycelia ofPholiota nameko, 18 monokaryotic stocks were used to produce a total of 130 dikaryotic stocks by reciprocal crossing. Monokaryotized mycelium was raised from dikaryotic mycelium in the peripheral zone of the growing colony. The stocks mated with a particular group of monokaryons produced wide-range monokaryotization at higher rates than the other combinations of hybridization. The growth rates of the monokaryotized mycelia exceeded from those of the corresponding parental dikaryons. The monokaryotized mycelium was isolated and back-crossed to parental monokaryotic stocks. Most of the isolates had nuclear types similar to only one of the parental stocks, while the replicates of isolates from two dikaryotic hybrids showed split nuclear type compositions. It is suggested that a relative dominance is active in the selection of one of the two nuclei of the dikaryotic cells in monokaryotization. The hierarchy of relative dominance among nuclei of 18 parental monokaryotic stocks in the monokaryotization of their reciprocal crossing products was estimated. We propose the involvement of a cascade process in dikaryotic cell division, in which the first dividing nucleus (to be found in the monokaryotized cell) may act as the leading nucleus and the other one as the following nucleus.     

Conjugational transfer systems of Rhodopseudomonas capsulata mediated by R plasmids

Plasmids RP1, R68.45 and RP4::Mu cts 61 were transferred into Rhodopseudomonas capsulata from Escherichia coli. The frequency of intraspecies transfer of these plasmids in R. capsulata was 10^-4–10^-5 per donor. The plasmids also mobilized chromosomal genes at a low frequency. Phototrophic recombinants from matings between recipient strains defective in the photosynthetic-apparatus and wild type donors were obtained at a frequency of 10^-7–10^-8 per donor.     

Kinetics of plasmid transfer among <Emphasis Type="Italic">Bacillus cereus </Emphasis>group strains within lepidopteran larvae

The cry toxin encoding plasmid pHT73 was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to six B. cereus group strains in three lepidopteran (Spodoptera exigua, Plutella xyllostella and Helicoverpa armigera) larvae by conjugation. The conjugation kinetics of the plasmid was precisely studied during the larval infection using a new protocol. The infections were performed with both vegetative and sporulated strains. However, larval death only occurred when infections were made with spore and toxin preparations. Likewise, spore germinations of both donor and recipient strains were only observed in killed larvae, 44–56 h post-infection. Accordingly, kinetics showed that gene transfer between B. thuringiensis strain KT0 and other B. cereus strains only took place in dead larvae among vegetatively growing bacteria. The conjugational transfer ratios varied among different strain combinations and different larvae. The highest transfer ratio reached 5.83 × 10⁻⁶ CFU/donor between the KT0 and the AW05R recipient in Helicoverpa armigera, and all transconjugants gained the ability to produce the insecticidal crystal. These results indicated that horizontal gene transfer among B. cereus group strains might play a key role for the acquisition of extra plasmids and evolution of these strains in toxin susceptible insect larvae.     

Factors controlling the loss of immunoreactive somatic histone H1 from blastomere nuclei in oocyte cytoplasm: a potential marker of nuclear reprogramming

Nuclei of differentiated cells can acquire totipotency following transfer into the cytoplasm of oocytes. While the molecular basis of this nuclear reprogramming remains unknown, the developmental potential of nuclear-transfer embryos is influenced by the cell-cycle stage of both donor and recipient. As somatic H1 becomes immunologically undetectable on bovine embryonic nuclei following transfer into ooplasm and reappears during development of the reconstructed embryo, suggesting that it may act as a marker of nuclear reprogramming, we investigated the link between cell-cycle state and depletion of immunoreactive H1 following nuclear transplantation. Blastomere nuclei at M-, G1-, or G2-phase were introduced into ooplasts at metaphase II, telophase II, or interphase, and the reconstructed embryos were processed for immunofluorescent detection of somatic histone H1. Immunoreactivity was lost more quickly from donor nuclei at metaphase than at G1 or G2. Regardless of the stage of the donor nucleus, immunoreactivity was lost most rapidly when the recipient cytoplast was at metaphase and most slowly when the recipient was at interphase. When the recipient oocyte was not enucleated, however, immunoreactive H1 remained in the donor nucleus. The phosphorylation inhibitors 6-DMAP, roscovitine, and H89 inhibited the depletion of immunoreactive H1 from G2, but not G1, donor nuclei. In addition, immunoreactive H1 was depleted from mouse blastomere nuclei following transfer into bovine oocytes. Finally, expression of the developmentally regulated gene, eIF-1A, but not of Gapdh, was extinguished in metaphase recipients but not in interphase recipients. These results indicate that evolutionarily conserved cell-cycle-regulated activities, nuclear elements, and phosphorylation-linked events participate in the depletion of immunoreactive histone H1 from blastomere nuclei transferred in oocyte cytoplasm and that this is linked to changes in gene expression in the transferred nucleus.     

Efficiency of donor cell preparation and recipient oocyte source for production of transgenic cloned dairy goats harboring human lactoferrin

The objective was to investigate the effects of the transgenic donor cell synchronization method, oocyte sources, and other factors, on production of hLF-gene nucleus transfer dairy goats. Three transfected cell lines from ear biopsies from three 3-mo-old Saanen dairy goats (designated Number 1, Number 2, and Number 3, respectively) were selected as karyoplast donors for somatic cell nuclear transfer (SCNT) after detailed identification (including PCR and sequencing of PCR products). In donor cell cycle synchronization studies, the apoptosis rate of hLF transgenic fibroblasts was not different (P > 0.05) after 3 days of serum starvation or 2 days of contact inhibition. Additionally, there was no effect (P > 0.05) on developmental capacity of reconstructed embryos; however, the kidding rate of recipients in the serum starvation group was higher than that in the contact inhibition group (18 vs. 0%, respectively). The production efficiency of the transgenic cloned goats using donor cells from the Number 1 dairy goat cell line was higher than those using the Number 2 and the Number 3 cell lines (kidding rates were 18, 2, and 0%, respectively, P < 0.05). The oocyte source did not significantly affect the pregnancy rate of hLF-transgenic cloned dairy goats, but more fetuses were aborted when using in vitro matured oocytes compared to in vivo matured oocytes. In summary, utilizing transfected 3-mo-old dairy goat fibroblasts as donor cells, seven live offspring were produced, and the hLF gene was successfully integrated. This study provided additional insights into preparation of donor cells and recipient oocytes for producing transgenic cloned goats through SCNT.     

ITS polymorphism within a single strain ofSclerotium rolfsii

Two morphologically distinct strains, 63–76 and 63H1, were isolated from a protoplast and a hyphal tip of the parentalSclerotium rolfsii strain S-63, respectively. Strains 63–76 and 63H1 showed reduced mycelial growth and lacked clamp connections on hyphae. The two strains also differed from each other and from their parent in RAPD patterns generated by several primers, suggesting that 63–76 and 63H1 were homokaryons isolated from the hetereokaryon S-63. Whereas the parent S-63 belonged to ITS-RFLP group 1, RFLP patterns of internal transcribed spacer (ITS) regions of rDNA of 63–76 and 63H1 were similar to those of ITS-RFLP groups 5 and 3, respectively. The sequence similarity of ITS regions were more than 99% between 63–76 and group 5 strains, 100% between 63H1 and the group 3 strain, and 96.3% between 63–76 and 63H1. Direct sequencing failed in the parental strain S-63. S-63 was considered to contain ITS types of groups 5 and 3.     

Isoenzyme diversity in Reynoutria (Polygonaceae) taxa: escape from sterility by hybridization

The genus Reynoutria is represented by four taxa in the Czech Republic – R. japonica var. japonica and compacta, R. sachalinensis and R. × bohemica. Using isoenzyme analysis, we determined the degree of genotype variability in all taxa and compared clones of R. japonica var. japonica from the Czech Republic with those from Great Britain. While the rarely occurring tetraploid variety R. japonica var. compacta possesses low variability, the octoploid female clone of R. japonica var. japonica is genetically uniform in the 93 clones sampled and belongs to the same genotype that is present in the whole Europe. R. japonica var. japonica can be fertilized by the pollen of tetraploid R. sachalinensis and a hexaploid hybrid R. × bohemica is produced. In R. sachalinensis, 16 genotypes were found in the 50 clones sampled. R. × bohemica is genetically the most diverse taxon in the study area, with 33 genotypes recorded among 88 clones sampled.     

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.     

Fate of transforming DNA following uptake by competent Bacillus subtilis

Summary During transformation in Bacillus subtilis, donor and recipient DNA are initially associated by non-covalent bonds. The donor and recipient moieties later become covalently joined. The molecular weight of the donor component, when freed from the noncovalent complex by sucrose gradient sedimentation under alkaline conditions, ranges from 1 to 5×10⁶, with an average of about 2.5 to 3.0×10⁶. The latter values are in good agreement with previous measurements of the size of the integrated donor fragment.     

Association between IL-4 polymorphism and acute rejection of solid organ allograft: A meta-analysis

Background

Cytokines have been implicated in the acute rejection of solid organ transplantation. Many studies have investigated the association between recipient or donor IL-4 polymorphism and acute rejection, with different studies reporting inconclusive results.

Methods

We searched PUBMED and EMBASE until June 2012 to identify eligible studies investigating the association between IL-4 polymorphism with acute rejection after solid organ transplantation. Statistical analysis was performed using STATA10.0.

Results

A total of 12 studies were included. Pooled ORs suggested 1) no significant association was detected between recipient or donor IL-4 − 590C/T polymorphism and acute rejection of solid allograft; 2) no significant association was detected between recipient IL-4 − 33C/T polymorphism and acute rejection of solid allograft; 3) when stratified by transplantation type, IL-4 − 590C/T polymorphism was associated with acute rejection of liver transplantation (T/T + C/T vs. C/C: OR = 0.36, 95%CI = 0.14–0.90); 4) significantly decreased risk of acute rejection was detected in recipient IL-4 − 590*T-negative/donor T-positive genotype pairs than all other recipient–donor IL-4 − 590T/C pairs (OR = 0.14, 95%CI = 0.03–0.66).

Conclusions

Our meta-analysis suggested that recipient IL-4 − 590C/T polymorphism was associated with acute rejection of liver transplantation, but nor renal or heart transplantation. It was also suggested that combined recipient IL-4 − 590*T-negative/donor T-positive genotype may suffer decreased risk of acute rejection of solid allograft. Further well-designed studies with larger sample size were required to verify our findings, with focus on the association of IL-4 polymorphism with acute rejection in patients with liver transplantation and studies investigating combined recipient–donor genotype.