Gave PCR Products Research Articles

Incidence of virulence genes in predominant Brucella strains among domestic animals in Egypt

To investigate the incidence of the virulence genes among predominant Brucella strains in infected cattle, buffaloes, sheep, goats, and camels as well as in humans in Egypt, a total of 263 samples (85 milk, 167 tissue, 11 whole blood samples), yielded 140 (53.2%) Brucella isolates. Confirmation of Brucella isolates was carried out by conventional biotyping and by PCR using IR1/IR2 primers targeting Brucella genus showing an amplicon of 839 bp in all Brucella isolates. Conventional bioty­ping, as well as duplex PCR of the isolated non-repetitive Brucella strains, identified 107 (76.4 %) as B. melitensis with an amplicon of 731 bp and 33 (23.6%) as B. abortus with an amplicon of 498 bp. Out of 87 Brucella strains isolated from cattle, 63 (72.4%) were B. melitensis. No Brucella isolates were obtained from 7 lymph nodes of camels or 11 human blood samples; however, DNA extraction from 7 human sera and 3 camel lymph nodes gave positive PCR yield. All these samples gave PCR products indicating infection with B. melitensis. The distribution of the virulence genes among 33 B. abortus isolates revealed that virB recorded the highest incidence (97%), then followed bvfA, ure, and omp25 (93.9%), wbkA (90.9%), manB (87.9%) and amiC (84.8%). All 107 B. melitensis isolates had the bvfA, virB, and omp25 genes, while the prevalence of ure was 99.1%, that of wbkA: 96.3%, manB: 95.3% and amiC: 94.4%. The obtained results indicated the high incidence of virulence genes among field Brucella strains among farm animals in Egypt.

The relationship between the sources of lactobacillus isolates and their antimicrobial activity

Lactobacillus genus was isolated from two different sources, healthy infants feces and some dairy products, identify isolates by traditional methods and confirm diagnosis by molecular detection. Susceptibility was tested to some antibiotic then supernatants antimicrobial activity were tested against some pathogenic bacteria involved Escherichia coli O157:H7, Pseudomonas aeruginosa, Enterococcus faecalis , Staphylococcus aureus, Klebsiella sp., Salmonella typhimiurum and Clostridium sp.. Morphological, Microscopically and Biochemical tests results showed that eleven isolates were lactobacillus, DNA was extracted from isolates and PCR reaction was performed to confirm diagnosis using a specific primer for 16SrDNA in Lactobacillus genus, Lacto F and Lacto R, all isolates gave PCR products with molecular weight 231bp. Antibiotic susceptibility test showed that Lactobacillus isolates of healthy infants feces were appeared multi resistant to more than one of antibiotics and sensitive to chloramphenicol while Lactobacillus isolates of dairy products were less resistance to antibiotics and most of them are sensitive to antibiotics , also results showed varying inhibitory effect of all Lactobacillus isolates supernatants in growth of pathogenic bacteria in this study, healthy infants feces isolates showed highest inhibition zone than dairy products Lactobacillus isolates.

Escherichia coli H-Genotyping PCR: a Complete and Practical Platform for Molecular H Typing.

In Escherichia coli, more than 180 O groups and 53 H types have been recognized. The O:H serotyping of E. coli strains is an effective method for identifying strains with pathogenic potential and classifying them into clonal groups. In particular, the serotyping of Shiga toxin-producing E. coli (STEC) strains provides valuable information to evaluate the routes, sources, and prevalence of agents in outbreak investigations and surveillance. Here, we present a complete and practical PCR-based H-typing system, E. coli H-genotyping PCR, consisting of 10 multiplex PCR kits with 51 single PCR primer pairs. Primers were designed based on a detailed comparative analysis of sequences from all H-antigen (flagellin)-encoding genes, fliC and its homologs. The specificity of this system was confirmed by using all H type reference strains. Additionally, 362 serotyped wild strains were also used to evaluate its practicality. All 277 H-type-identified isolates gave PCR products that corresponded to the results of serological H typing. Moreover, 76 nonmotile and nine untypeable strains could be successfully subtyped into any H type by the PCR system. The E. coli H-genotyping PCR developed here allows broader, rapid, and low-cost subtyping of H types and will assist epidemiological studies as well as surveillance of pathogenic E. coli.

Studies on Vibriosis among Some Marine Fishes in Lake Temsah

During the current study, a total of 160 marine fishes of two different species represented as: seabass (Dicentrarchus labrax) and Tilapia zillii randomly collected from Lake Temsah at Ismailia governorate from August 2015 to July 2016. Seventy-six of fishes found to be infected with both V. anguillarum and V. parahemolyticus, as Gram negative, short rod bacilli as comma shaped and motile bacteria. D. labrax was the most infected fish spp 41 %, pursued by Tilapia zillii 35 %. The highest prevalence of vibriosis among naturally infected marine fishes was recorded during summer 67.5 %, pursued by spring 55 %, then autumn 40 %, while the minimal prevalence 27.5 % was registered during winter. Molecular investigation of pure isolates using sets of both universal and specific primers gave PCR products with clear band of 120 and 519 bp lengths, respectively. Experimentally infected fish subjected to a dose (107) of V. anguillarum presented higher morbidity and mortality rate up to 80 %. Most of infected fish showed skin ulceration in the ocular side, others displayed redness and scattered hemorrhagic spots on the abdomen with fin erosion. The histopathology of naturally infected marine fishes exhibited severe inflammatory reactions manifested by leukocytes infiltration with activation of melanomacrophage centers as well as tissue necrosis.

Diversity and genetic structure of Ornithogalum L. (Hyacinthaceae) populations as revealed by RAPD-PCR markers

Random amplified polymorphic DNA (RAPD) PCR method was used to assess the level of diversity and genetic structure in Ornithogalum L. populations from Serbia and Hungary with the main goal of improving the knowledge of this genus in the given region. The material was collected from 19 populations and identified as two morphologically similar and phylogenetically close taxa: O. umbellatum L. 1753 and O. divergens Boreau 1887. All ten RAPD primers used for the analysis gave PCR products, with length between 3000bp and 300bp. There were 101 amplified fragments in total; number of polymorphic bands per primer varied between seven and 13. Percentage of polymorphic loci was 96% in total and 12% in average in each population. Genetic variation statistics for all loci also showed that genetic diversity for all populations was 0.29 and Shannon index 0.45, while mean values for these parameters calculated for each population were 0.04 and 0.06, respectively. Analysis of molecular variance demonstrated high population genetic differentiation; however Mantel test showed no significant correlation between geographic distances of populations and genetic distances expressed through population pairwise FST. UPGMA dendrogram based on Jaccard genetic similarity coefficients showed subclustering and principal coordinate analysis based on Nei and Li coefficients of genetic distances indicated grouping. Analysis of populations genetic structure was in accordance with these results and clearly separated populations of O. umbellatum from O. divergens. RAPDs proved to be a reliable and rapid method suitable for distinguishing genetic differentiation in Ornithogalum, thus could be applied as a useful additional tool in resolving taxonomic problems.

First Report of Alternaria alternata Causing Fruit Rot on Fig (Ficus carica) in Montenegro.

In July 2012, a fruit rot disease was observed in several commercial fig tree orchards located in the Podgorica region in Montenegro. Symptoms on fruits initially appeared as small circular to oval, light brown, necrotic, sunken spots located mostly on the areas surrounding the ostiolar canal with an average diameter of 5 to 10 mm, which gradually enlarged in size leading to total fruit rot. Disease incidence on fruit across the fields ranged from 15 to 20% but the disease did not increase further due to hot and dry conditions thereafter. No foliar symptoms were observed. Small pieces (5 mm2) of symptomatic fruits were excised from the junction of diseased and healthy tissue, surface sterilized in 70% ethanol solution for 1 min, washed in three changes of sterile distilled water, air dried, and transferred to potato dextrose agar (PDA). After 2 to 3 days of incubation at 25°C, a fungus was consistently isolated. The isolates had radial growth and produced sooty black colonies. Microscopic observations of the colonies revealed brown septate hyphae and simple or branched conidiophores 30 to 65 μm long and 3 to 4.5 μm wide. Dark brown conidia were in chains (3 to 7), sized 10 to 35 × 5 to 9 μm, ellipsoid to ovoid, with 2 to 5 transverse and a few (1 to 3) to no longitudinal septa. Based on morphological characteristics, the fungus was identified as Alternaria alternata (3). For molecular identification, DNA was extracted from mycelia and conidia of two representative single spore isolates designated as ALT1-fCG and ALT2-fCG. PCR was carried out using internal transcribed spacer (ITS) region primers ITS4/ITS5 and A. alternata species-specific primers AAF2/AAR3 (1). Both primer pairs gave PCR products that were subjected to direct sequencing. BLAST analysis of the 546-bp ITS4/ITS5 (KF438091) and 294-bp AAF2/AAR3 (KF438092) sequences revealed 100% identity with several A. alternata isolates. Pathogenicity tests were conducted on 30 detached almost ripe and healthy fig fruit (cv. Primorka) by spraying them with a conidial suspension of the isolated fungus (106 conidia/ml) with a handheld sprayer. Thirty fruit inoculated with sterile water served as the non-inoculated control. Inoculated and control fruit were kept in a moist chamber at 25°C. Symptoms appeared on inoculated fruit 2 to 3 days after inoculation and all fruit were completely rotted 5 to 6 days after inoculation. Control fruit did not display any symptoms. A. alternata was consistently re-isolated from inoculated fruit, fulfilling Koch's postulates. The fig fruit rot caused by A. alternata has been reported before in California (2) and elsewhere mainly as postharvest pathogen. To our knowledge, this is the first report of fruit rot caused by A. alternata on fig in Montenegro. Considering Podgorica as the largest fig-producing area and the importance of fig as a traditionally grown crop, it could pose a threat to fig production in Montenegro. Voucher specimens are available at the culture collections of the University of Montenegro, Biotechnical Faculty.

Targeted Gene Addition in Human Epithelial Stem Cells by Zinc-finger Nuclease-mediated Homologous Recombination

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a "safe harbor" locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of > 20% in a human keratinocyte cell line, > 10% in immortalized keratinocytes, and < 1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs.

Validation and comparison of microsatellite markers derived from Senegalese sole (Solea senegalensis, Kaup) genomic and expressed sequence tags libraries

In this work, we tested 100 potential new microsatellites (SSRs) equally derived from expressed sequence tag (EST) and enriched genomic-DNA libraries from Senegalese sole (Solea senegalensis, Kaup), a valuable cultured flatfish species. A final set of 69 new polymorphic microsatellites were validated after a population analysis, 37 of which corresponded to the first EST library constructed for Senegalese sole (EST-SSR). Although differences were not significant, EST sequences provided a higher proportion of quality markers (74%) than anonymous ones (64%). Most of the rejected anonymous SSRs (17 loci) were discarded because they did not generate PCR products; only one was monomorphic. On the contrary, all EST-SSRs gave PCR products, although monomorphism was more frequent (26%). Altogether, the number of alleles per locus was fairly similar in both SSR types, ranging from 2 to 19. The observed and expected heterozygosities varied from 0.105 to 1 and from 0.108 to 0.937, respectively. The main difference between the two sets was the percentage of annotated loci, being higher in EST-SSRs, as expected. Within the EST-SSRs, 46% of them showed flanking regions that significantly matched with EST sequences from other three flatfish species; however, the microsatellite itself was present only on half of these cases. These two new SSR sets constitute a suitable tool for fingerprinting, gene flow, genetic diversity, genome mapping studies and molecular-assisted breeding in this species.

Exogenous isolation of conjugative plasmids from pesticide contaminated soil

Exogenous plasmid isolation method was used to assess conjugative plasmids conferring pesticide tolerance/multiple metal and antibiotic resistance from contaminated soil using bacteria detached from soil samples as a donor and rifampicin resistant E. coli HMS as a recipient strain on mineral salt agar medium supplemented with γ-HCH, and antibiotics ampicillin, tetracycline, chloramphenicol and kanamycin. Transconjugants were obtained on ampicillin (10μg/ml) and tetracycline (20μg/ml) amended MSA plates and frequency of ampicillin and tetracycline resistance gene transfer was 7.2×10(-6) and 9.2×10(-4) transconjugants/recipient, respectively. PCR typing methods were used to assess the presence of plasmids of the incompatibility groups IncP, IncN, IncW, IncQ and rolling circle plasmids of pMV158 type in DNA derived from transconjugants. All transconjugants were PCR amplified for the detection of Inc group plasmids and rolling circle plasmids of pMV158 family in which TM2, 3, 4, 11 and 12 (tet) transconjugants gave PCR products with the IncP-specific primers for both replication and transfer functions (trfA2 (IncP) and oriT (IncP)), while TM 14 (amp) gave an IncP specific PCR product for the replication gene trfA2 (IncP) only. TM15, 16, 18 and 21 (amp) gave a PCR product for the IncW-specific oriT (IncW). Out of 24 transconjugants, only TM 5 (tet) gave a PCR product with the pMV158 specific primer pair for oriT (RC). Our findings indicate that Inc group plasmids and rolling circle plasmids of pMV158 type may be responsible for transferring multiple antibiotic resistance genes among the bacterial soil community.

In silico mining and characterization of simple sequence repeats from gilthead sea bream ( Sparus aurata) expressed sequence tags (EST-SSRs); PCR amplification, polymorphism evaluation and multiplexing and cross-species assays

We screened for simple sequence repeats (SSRs) found in ESTs derived from an EST-database development project (‘Marine Genomics Europe’ Network of Excellence). Different motifs of di-, tri-, tetra-, penta- and hexanucleotide SSRs were evaluated for variation in length and position in the expressed sequences, relative abundance and distribution in gilthead sea bream ( Sparus aurata). We found 899 ESTs that harbor 997 SSRs (4.94%). On average, one SSR was found per 2.95 kb of EST sequence and the dinucleotide SSRs are the most abundant accounting for 47.6% of the total number. EST-SSRs were used as template for primer design. 664 primer pairs could be successfully identified and a subset of 206 pairs of primers was synthesized, PCR-tested and visualized on ethidium bromide stained agarose gels. The main objective was to further assess the potential of EST-SSRs as informative markers and investigate their cross-species amplification in sixteen teleost fish species: seven sparid species and nine other species from different families. Approximately 78% of the primer pairs gave PCR products of expected size in gilthead sea bream, and as expected, the rate of successful amplification of sea bream EST-SSRs was higher in sparids, lower in other perciforms and even lower in species of the Clupeiform and Gadiform orders. We finally determined the polymorphism and the heterozygosity of 63 markers in a wild gilthead sea bream population; fifty-eight loci were found to be polymorphic with the expected heterozygosity and the number of alleles ranging from 0.089 to 0.946 and from 2 to 27, respectively. These tools and markers are expected to enhance the available genetic linkage map in gilthead sea bream, to assist comparative mapping and genome analyses for this species and further with other model fish species and finally to help advance genetic analysis for cultivated and wild populations and accelerate breeding programs.

New analysis of EST-SSR distribution and development of EST-SSR markers in Salvia miltiorrhiza

To establish the new EST-SSR markers for analyzing the genetic variation of different population of Salvia miltiorrhiza. It was dealt with ESTs newly downloaded from Genbank and that of acquired from HMPL lab EGassembler software, and then carried out SSR loci search and SSR type analysis by SSRIT software. After that, it was designed the EST-SSR primer pairs for PCR amplification condition optimization. Abundant and high coverage of SSR loci distribution were found in S. miltiorrhiza with having one SSR per 5.8 kb ESTs. Among them, the occurrences of different repeat units were mainly the di- (63.0%) and tri- (35.5%). The CT/AG was the most frequent motif in dinucleotide motif type and the GAA/TCC was the most frequent motif in trinucleotide repeats. Out off 36 primer pairs, 29 primer pairs (80.5%) were successfully amplified in all samples of S. miltiorrhiza while the rest failed to give PCR products at various annealing temperature and Mg2+ concentrations. The selected primer pairs also showed the polymorphism in samples from different S. miltiorrhiza populations. The newly establishment of EST-SSR markers showed high SSR loci coverage and genetic polymorphisms in S. miltiorrhiza population. It could be used for genetic variation analysis.

DETECTION OF GENETICALLY MODIFIED SOYBEANS DNA IN A CHEESE LIKE PRODUCT AND SOME HEAT-TREATED PRODUCTS AS FOOD MODEL

This work was conduct to detect of recombinant DNA of genetically modified (GM) in soybean like cheese and heat-treated GM soybean milk. Genomic DNA was extracted from soybean like cheese and heat-treated GM soybeans milk by using the cetyltrimethylammonium bromide (CTAB) method and vivants DNA extraction kit. Primer pair P35s-f2 /petu-r1 amplify the junction region between CaMV35S promoter and part of CP4EPSPS and GMOScreen kit that amplify CaMV35S promoter and or NOS terminator were selected to detect the recombinant of DNA in Roundup Ready soybean. They gave PCR products of band sizes, 172 and 123 bp. PCR products were detected when DNA solution extracted from soybean like cheese and heat-treated GM soybean milk by the vivants DNA extraction kit. PCR products of the expected 172, and 123 bp were detected by agarose gel electrophoresis at the end of storage period 60 days. However, PCR products were not detected in DNA extracted from soybean like cheese and heat-treated GM soybean milk by the CTAB method. These results indicate that judicious selection of DNA extraction methods and target sequences are important to detect DNA from soybean like cheese and recombinant DNA can be detected in soybean like cheese

Canis familiaris telomerase reverse transcriptase undergoes alternative splicing

The enzyme telomerase is essential for cell proliferation and tumorigenesis. Telomerase reverse transcriptase (TERT) represents the catalytic subunit of the enzyme. In humans, TERT expression is regulated by several different mechanisms, including alternative splicing. Canis familiaris TERT (dogTERT) has been shown to have a high level of sequence similarity with human TERT, indicating that the dog may represent a suitable animal model for telomerase studies. In the present report we sought to investigate whether dogTERT undergoes alternative splicing. During the analysis of canine mammary tissues (both tumor and paired adjacent to the tumor normal tissues) for dogTERT expression by RT-PCR, we identified eight samples-one tumor and seven adjacent normal-which gave PCR products of unexpected sizes. DNA sequencing revealed two insertions (175 and 28 bp long) and two deletions (17 and 32 bp long), which were encountered in different combinations and gave rise to five different transcripts. The generation of all variants could be explained by the employment of alternative splicing sites within dogTERT genomic sequences. The 175-bp and 28-bp insertions, identified between exons 7 and 8 and between 8 and 9, respectively, constituted unspliced sequences of introns 7 and 8, respectively. Both deletions originated from exon 8 sequence removals due to alternative splicing. All five variants encoded truncated proteins, which lacked essential motifs for reverse transcription and might have thus lost their ability to compose active telomerase enzymes. This is the first identification of alternative splicing events within dogTERT. The results presented here may provide the basis for more thorough studies on the regulation of telomerase activity in canine normal and cancer cells.

Low-Intensity Exercise Exerts Beneficial Effects on Plasma Lipids via PPARγ

An important mechanism by which physical activity reduces the risk of cardiovascular disease is through regulating plasma lipids. We investigated whether low-intensity exercise modulates lipid metabolism and the transcription factors peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor alpha (LXRalpha) responsible for controlling reverse cholesterol transport (RCT). Thirty-four sedentary adults, mean age 45.6 +/- 11.1 yr, participated in an 8-wk low-intensity exercise program consisting of walking 10,000 steps, three times a week. Subjects were randomly allocated to either an exercise group or a sedentary control group, and serum lipid or lipoprotein concentrations were determined. Compared with controls, there was a significant decrease in total cholesterol (preexercise, 5.73 +/- 1.39 mmol x L; postexercise, 5.32 +/- 1.28 mmol x L) and a significant increase in HDL (preexercise, 1.46 +/- 0.47 mmol x L; postexercise, 1.56 +/- 0.50 mmol x L) after the exercise program. There was a significant increase in serum oxidized LDL (oxLDL) concentrations in the exercise group before and after exercise (0 wk, 554 +/- 107 ng x mL; 4 wk, 698 +/- 134 ng x mL; 8 wk, 588 +/- 145 ng x mL). A significant increase in leukocyte mRNA expression for PPARgamma (4 wk, 1.8 +/- 0.9-fold; 8 wk, 4.3 +/- 1.9-fold) was observed, which was reinforced by increased PPARgamma DNA-binding activity postexercise (preexercise, 0.22 +/- 0.09 OD units; postexercise, 1.13 +/- 0.29 OD units). A significant increase in gene expression was observed for the oxLDL scavenger receptor CD36 (4 wk, 3.8 +/- 0.6-fold; 8 wk, 2.7 +/- 0.5-fold) and LXRalpha (8 wk, 3.5 +/- 0.8-fold). Two LXRalpha-regulated genes involved in RCT, namely, ATP-binding cassette transporters A1 and GI (ABCA1 and ABCG1, respectively), were significantly up-regulated postexercise (8 wk: ABCA1, 3.46 +/- 0.56-fold; ABCG1, 3.06 +/- 0.47-fold). We propose that the net effect of these changes may be to increase oxLDL uptake, to stimulate RCT, and thus to promote clearance of proatherogenic lipids from the vasculature, ultimately contributing to the cardiovascular benefits of low-intensity aerobic exercise.

Occurrence in Tunisia of potato tuber necrotic ringspot disease (PTNRD) caused by variant PVYNTNof Potato virus Y

Potato tuber necrotic ringspot disease (PTNRD) (Beczner et al., 1984) is now found worldwide. The causal agent has been characterised as a variant of Potato virus Y (PVY), designated PVYNTN. Most PVYNTN isolates display a recombinant genome with exchanges of sequences in the HC-Pro, NIa and CP genes between PVYN and PVYO (Glais et al., 2002; Nie & Singh, 2003). In 2005, PTNRD symptoms were observed in autumn crops in southern Tunisia. Many harvested tubers displayed superficial necrotic ring spots. Plantlets grown from a collection of these tubers tested positive for PVY when tested by ELISA using an anti-PVY antiserum (Inra-Fnpppt, Le Rheu, France). Eleven isolates were aphid transmitted from potato plantlets onto Nicotiana tabacum cv. Xanthi. Isolates from tobacco were compared to well-characterized isolates representative of PVY strains and variants: PVYN, PVYNTN, PVYNW and PVYO (Glais et al., 2002). All isolates were maintained in a regulated (20°C ± 2°C), insect-proof greenhouse. All Tunisian isolates tested in tobacco gave leaf distortion and vein necrosis, as did reference isolates of the three PVYN subgroups. Tunisian isolates also reacted positively in ELISA with specific anti-PVYN antibodies (Inra-Fnpppt) and negatively with anti-PVYO-C antibodies (Adgen). Hence biological and serological typing showed that none of the Tunisian isolates belonged to the PVYNW variant since PVYNW isolates are known to induce necrosis on tobacco and to react negatively with anti-PVYN antibodies. Four Tunisian isolates were characterized at the molecular level using RT-PCR protocols with specific primers, either based on the polymorphism in the P1 gene (Glais et al., 2001), or targeting the three recombination sites specific to PVYNTN isolates, RJ1 and RJ2 (Nie & Singh, 2003), RJ3 (L. Glais, unpublished data). Tunisian isolates gave PCR products with sizes identical to those produced by the reference PVYNTN isolates, which demonstrated they are PVYNTN isolates with recombination events in HC-Pro/P3, CI/NIa and C-ter CP regions. This is the first report of PVYNTN isolates associated with PTNRD in Tunisia. PTNRD disease has never been previously documented in any potato growing area in Tunisia. Further studies are in progress to assess the incidence of the PVYNTN variant in both spring and autumn potato crops. The authors would like to thank A. Najjar and M. Guillet for their excellent assistance and R. Kutcher for his help with English.

Conjugative plasmids in multi-resistant bacterial isolates from Indian soil

Determination of heavy metal and antibiotic resistance and presence of conjugative plasmids in bacteria isolated from soil irrigated with wastewater. Composite soil samples were collected from Ghaziabad, Uttar Pradesh, India. Forty different bacteria were selected from nutrient agar and characterized by morphological, cultural and biochemical tests. All the isolates were tested for their resistance to different heavy metals and antibiotics. The DNA derived from multiple metal and antibiotic-resistant bacterial isolates was PCR amplified and plasmid-specific sequences (IncP, IncN, IncW, IncQ and pMV158-type) were analysed by dot blot hybridization. All isolates gave PCR products with trfA2 and oriT primers of the IncP group. These PCR products also hybridized with the RP4-derived probes. However, the samples were negative for all the other investigated plasmids as proved by PCR and dot blots. The presence of conjugative/mobilizable IncP plasmids in the isolates indicates that these bacteria have gene-mobilizing capacity with implications for potential dissemination of introduced recombinant DNA. The detection of IncP plasmids in all the bacterial isolates is another proof for the prevalence of these plasmids. We propose that IncP plasmids are mainly responsible for the spread of multi-resistant bacteria in these soils.

Development of a multilocus sequence typing scheme for intestinal spirochaetes within the genus Brachyspira

The purpose of this study was to evaluate a multilocus sequence typing (MLST) scheme for intestinal spirochaetes of the genus Brachyspira. Eight loci mainly coding for enzymes previously used in multilocus enzyme electrophoresis analysis of Brachyspira species were examined in 66 Brachyspira field isolates and type/reference strains. The isolates and strains were recovered from pigs, birds, dogs and a mouse and originated from seven European countries, the USA and Canada. Forty-six isolates represented recognized Brachyspira species and 20 represented provisionally designated species or isolates that have not been classified. Only two loci gave PCR products for all 66 strains and isolates, but amplicons for seven loci were obtained for 44 of the isolates. Sequences for each locus had a DNA allelic variation of 30-47 and an amino acid allelic variation of 14-47 that gave rise to the same number of sequence and amino acid types (58) for the strains and isolates studied. A population snapshot based on sequence and amino acid types showed a close phylogenetic relationship amongst the porcine isolates from the same geographical regions, and indicated a close evolutionary relationship between isolates recovered from pigs and mallards. A general concordance was obtained between the MLST groupings and classifications based on culture and biochemical tests, 16S rDNA sequence analysis and random amplified polymorphic DNA analysis. This is a first step towards establishing an MLST system for use in identifying Brachyspira species and determining relationships between individual strains and species in the genus.

Characterization of Lepidoptera-active cry and vip genes in Iranian Bacillus thuringiensis strain collection

The characterization of 70 Bacillus thuringiensis ( Bt) strains isolated from different agro-ecological regions of Iran is presented. Characterization was based on PCR analysis using 25 general and specific primers for cry1, cry2, cry9 and vip3Aa genes encoding proteins active against Lepidoptera, crystal morphology, plasmid profiles, and protein band patterns as well as their insecticidal activity on Heliotis armigera. Isolates containing vip3 Aa gene were the most abundant (82.6%) followed by those containing cry2 (56.5%), cry1 (49%) and cry9 (30%). Twenty-two distinct cry1-type profiles were identified from only cry1-harboring isolates when these were analyzed with specific primers. Several of them were found to be different from all previously published profiles. Finally 7.24% of the isolates did not produce any PCR product. Some strains were positive by universal primers but negative by specific primers for all known genes of cry1, cry2 and cry9 or gave PCR products of different sizes when assayed with cry1C, cry1E, cry1J, cry9A, and vip3a specific primers. These strains may contain a new gene or genes that seem promising for biological control of insects and management of resistance. Based on morphological and molecular studies, 20 potentially Lepidopteran-specific active isolates were selected for bioassays. Four strains showed similar or higher activity against H. armigera larvae than Bt subsp. kurstaki ( Btk) and displayed high similarities with the Btk used in this study with regard to protein and plasmid profiles and the presence of cry genes. These results are important for directing future exploration of microbial control strategies for control of crop pests in the region.

Development of a microsatellite marker set applicable to genome-wide screening of cynomolgus monkeys (Macaca fascicularis)

To develop a microsatellite marker set applicable to genome-wide screening of cynomolgus monkeys (Macaca fascicularis), 148 microsatellite markers were selected from the human genome database. The polymorphisms and inheritance of PCR products were determined by screening twenty unrelated monkeys and by analysis of three families, respectively. As a result, 106 primers (72%) gave PCR products of the size expected for humans and rhesus monkeys. Among these products, polymorphism and single-gene inheritance in cynomolgus monkeys was observed for 66 markers (62%). The average number of alleles at the 66 polymorphic loci was 5.86 (range 2-10), and average heterozygosity was 0.63 (range 0.10-0.88). This is the first report of microsatellite markers for cynomolgus monkeys. Chromosomal mapping of these markers is now in progress.

Isolation of a coronavirus from a green-cheeked Amazon parrot (Amazon viridigenalis Cassin)

A virus (AV71/99) was isolated from a green-cheeked Amazon parrot by propagation and passage in both primary embryo liver cells derived from blue and yellow macaw (Ara ararauna) embryos and chicken embryo liver cells. Electron microscopic examination of cytopathic agents derived from both types of cell cultures suggested that it was a coronavirus. This was confirmed using a pan-coronavirus reverse transcriptase polymerase chain reaction that amplified part of gene 1 that encodes the RNA-dependent RNA polymerase. The deduced sequence of 66 amino acids had 66 to 74% amino acid identity with the corresponding sequence of coronaviruses in groups 1, 2 and 3. Several other oligonucleotide primer pairs that give PCR products corresponding to genes 3, 5, N and the 3′-untranslated region of infectious bronchitis virus, turkey coronavirus and pheasant coronavirus (all in group 3) failed to do so with RNA from the parrot coronavirus. This is the first demonstration of a coronavirus in a psittacine species. Isolement d'un coronavirus d'un perroquet amazone à joues vertes (Amazon viridigenalis Cassin) Un virus (AV71/99) a été isolé d'un perroquet amazone à joues vertes sur des cultures primaires d'hépatocytes d'embryon d'ara bleu (Ara ararauna) et d'hépatocytes d'embryon de poulet, en réalisant des passages. L'examen en microscopie électronique des agents cytopathogènes dérivés des deux types de culture cellulaire a suggéré la présence d'un coronavirus. Ceci a été confirmé en utilisant les réactions de transcription inverse et d'amplification en chaîne par polymérase qui ont amplifié une partie du gène 1 qui code l'ARN polymérase ARN dépendante. La séquence déduite de 66 acides aminés présentait 66 à 74% d'identité en acides aminés avec la séquence correspondante des coronavirus des groupes 1, 2, et 3. Plusieurs autres paires d'amorce oligonucléotidique qui donnent des produits PCR correspondant aux gènes 3, 5, N et à la région 3' non traduite du virus de la bronchite infectieuse, du coronavirus de la dinde et du coronavirus du faisan (tous du groupe 3) n'ont pu le faire avec l'ARN du coronavirus du perroquet. C'est la première mise en évidence d'un coronavirus chez les psittacidés. Isolierung eines Coronavirus aus einer Grünwangen-Amazone (Amazona viridigenalis Cassin) Aus einer Grünwangen-Amazone (Amazon viridigenalis Cassin) wurde durch Anzüchtung und Passagierung in primären Embryoleberzellen von blauen und gelben Aras (Ara ararauna) sowie in Hühnerembryoleberzellen ein Virus isoliert (AV71/99). Die elektronenmikroskopische Untersuchung der zytopathogenen Agens aus beiden Zellkulturtypen ließ vermuten, dass es sich um ein Coronavirus handelte. Dieses wurde mittels einer für alle Coronaviren spezifischen Reverse Transkriptase- Polymerasekettenreaktion, die Teile des für die RNS-abhängige RNS-Polymerase kodierende Gen 1 amplifizierte. Die abgeleitete Sequenz aus 66 Aminosäuren wies eine 66–74%ige Aminosäurenübereinstimmung mit den entsprechenden Sequenzen der Coronaviren der Gruppen 1, 2 und 3 auf. Mehrere andere Oligonukleotidprimerpaare, die den Genen 3, 5, N und der nicht translatierten 3‘-Region des infektiösen Bronchitisvirus, des Putencoronavirus und des Fasanencoronavirus (alle in Gruppe 3) entsprechende PCR-Produkte amplifizieren, gelang dies nicht mit der RNS aus dem Papageiencoronavirus. Dies ist die Erstbeschreibung eines Coronavirus in einer Psittazidenspezies. Aislamiento de un coronavirus de un loro Amapola (Amazon viridigenalis Cassin) Se aisló un virus (AV71/99) de un loro Amapola mediante propagación y pases en células primarias de hígado de embrión derivadas de embriones de guacamayo azul y amarillo (Ara ararauna) y en células hepáticas de embrión de pollo. Los estudios en microscopio electrónico de los agentes citopáticos procedentes de ambos tipos de cultivos celulares sugirieron que se trataba de un coronavirus. Esto se confirmó mediante una transcriptasa reversa-reacción en cadena de la polimerasa de pan-coronavirus que amplificaba parte del gen 1 que codifica la RNA polimerasa-RNA dependiente. La secuencia deducida de 66 aminoácidos tenía entre el 66 al 74% de similitud aminoacídica con la correspondiente secuencia de los grupos 1, 2 y 3 de Coronavirus. Otros pares de cebadores oligonucleotídicos, que producen productos de PCR correspondientes a los genes 3, 5, N y a la región no codificantes 3’ del virus de la bronquitis infecciosa, del coronavirus del pavo y del coronavirus del faisán (todos ellos en el grupo 3), no amplificaron en el caso del coronavirus del loro. Esta es la primera descripción de un coronavirus en especies de psitácidas.