Study of linkage between miniature and singed genes in Drosophila melanogaster

We have developed a practical exercise for undergraduate students whose main aim is to identify, using genetic crosses, a pair of D. melanogaster mutations (miniature and singed). Each student receives a vial with the problem strain containing two unknown mutations. The first step is to observe and describe both mutations. Then, the students carry out genetic crosses between mutant and normal strains: (P) ♀ mutant strain × ♂ normal strain (P) ♀ normal strain × ♂ mutant strain A different offspring is expected in these crosses: in the first one we will obtain normal females and m sn males, whereas in the second all individuals will present normal phenotype. It is possible to deduce that both are sex linked mutations. With this information and to simplify the amount of work, only F1 individuals from the first cross will be used (m+sn+ / m sn × m sn / Y chrom.) to obtain the F2 generation. By counting the number of miniature (recombinant type), singed (recombinant type), miniature-singed (parental type) and normal (parental type) flies it is possible to estimate the recombination frequency between both genes. Knowing the phenotype, their chromosomal location (X chromosome) and the genetic distance between both mutations, it is possible to identify them by finding all this information in a Drosophila melanogaster genetic map. Additionally, a statistical analysis can be carried out to compare the number of expected F2 individuals with those observed in the experiment. As the distance between both genes is 15.1 m.u., then the expected percentages for each phenotype would be: normal (42.45%), miniature-signed (42.45%), miniature (7.55%) and singed (7.55%). Multiplying the frequency of each class by the total number of individuals obtained in the F2 it is possible to estimate the expected number of flies for each class. Finally, a χ2 test can be computed to ascertain whether there are significant differences between expected and observed number of individuals.

Experimental Identification of Actinobacillus pleuropneumoniae Strains L20 and JL03 Heptosyltransferases, Evidence for a New Heptosyltransferase Signature Sequence.

We experimentally identified the activities of six predicted heptosyltransferases in Actinobacillus pleuropneumoniae genome serotype 5b strain L20 and serotype 3 strain JL03. The initial identification was based on a bioinformatic analysis of the amino acid similarity between these putative heptosyltrasferases with others of known function from enteric bacteria and Aeromonas. The putative functions of all the Actinobacillus pleuropneumoniae heptosyltrasferases were determined by using surrogate LPS acceptor molecules from well-defined A. hydrophyla AH-3 and A. salmonicida A450 mutants. Our results show that heptosyltransferases APL_0981 and APJL_1001 are responsible for the transfer of the terminal outer core D-glycero-D-manno-heptose (D,D-Hep) residue although they are not currently included in the CAZY glycosyltransferase 9 family. The WahF heptosyltransferase group signature sequence [S(T/S)(GA)XXH] differs from the heptosyltransferases consensus signature sequence [D(TS)(GA)XXH], because of the substitution of D(261) for S(261), being unique.

Genotypic comparison of Pantoea agglomerans plant and clinical strains

Pantoea agglomerans strains are among the most promising biocontrol agents for avariety of bacterial and fungal plant diseases, particularly fire blight of apple and pear. However, commercial registration of P. agglomerans biocontrol products is hampered because this species is currently listed as a biosafety level 2 (BL2) organism due to clinical reports as an opportunistichuman pathogen. This study compares plant-origin and clinical strains in a search for discriminating genotypic/phenotypic markers using multi-locus phylogenetic analysis and fluorescent amplified fragment length polymorphisms (fAFLP) fingerprinting.Results: Majority of the clinical isolates from culture collections were found to be improperly designated as P. agglomerans after sequence analysis. The frequent taxonomic rearrangements underwent by the Enterobacter agglomerans/Erwinia herbicola complex may be a major problem in assessing clinical associations within P. agglomerans. In the P. agglomerans sensu stricto (in the stricter sense) group, there was no discrete clustering of clinical/biocontrol strains and no marker was identified that was uniquely associated to clinical strains. A putative biocontrol-specific fAFLP marker was identified only in biocontrol strains. The partial ORF located in this band corresponded to an ABC transporter that was found in all P. agglomerans strains. Conclusion: Taxonomic mischaracterization was identified as a major problem with P.agglomerans, and current techniques removed a majority of clinical strains from this species. Although clear discrimination between P. agglomerans plant and clinical strains was not obtained with phylogenetic analysis, a single marker characteristic of biocontrol strains was identified whichmay be of use in strain biosafety determinations. In addition, the lack of Koch's postulate fulfilment, rare retention of clinical strains for subsequent confirmation, and the polymicrobial nature of P. agglomerans clinical reports should be considered in biosafety assessment of beneficial strains in this species

Molecular characterization of Brazilian strains of Xanthomonas campestris pv. viticola by rep-PCR fingerprinting

Bacterial canker of grapevine (Vitis vinifera), caused by Xanthomonas campestris pv. viticola was first detected in Brazil in 1998, affecting grapevines in the São Francisco river basin, state of Pernambuco. The disease was also reported in Juazeiro, Bahia and later in Piauí and Ceará. Due to its limited geographical distribution and relatively recent detection in Brazil, very little is known about the pathogen's biology and diversity. Repetitive DNA based-PCR (rep-PCR) profiles were generated from purified bacterial DNA of 40 field strains of X. campestris pv. viticola, collected between 1998 and 2001 in the states of Pernambuco, Bahia and Piauí. Combined analysis of the PCR patterns obtained with primers REP, ERIC and BOX, showed a high degree of similarity among Brazilian strains and the Indian type strain NCPPB 2475. Similar genomic patterns with several diagnostic bands, present in all strains, could be detected. Fingerprints were distinct from those of strains representing other pathovars and from a yellow non-pathogenic isolate from grape leaves. The polymorphism observed among the Brazilian strains allowed their separation into five subgroups, although with no correlation with cultivar of origin, geographic location or year collected.

Development of a scar marker for Pierce's Disease strains of Xylella fastidiosa

The objective of this research was to develop a primer for a polymerase chain reaction specific for Xylella fastidiosa strains that cause Pierce's Disease (PD) in grapes (Vitis vinifera). The DNA amplification of 23 different strains of X. fastidiosa, using a set of primers REP1-R (5'-IIIICGICGIATCCIGGC-3') and REP 2 (5'-ICGICTTATCIGGCCTAC-3') using the following program: 94 ºC/2 min; 35 X (94 ºC/1 min, 45 ºC/1 min and 72 ºC/1 min and 30 s) 72 ºC/5 min, produced a fragment of 630 bp that differentiated the strains that cause disease in grapes from the other strains. However, REP banding patterns could not be considered reliable for detection because the REP1-R and REP 2 primers correspond to repetitive sequences, which are found throughout the bacterial genome. The amplified product of 630 bp was eluted from the agarose gel, purified and sequenced. The nucleotide sequence information was used to identify and synthesize an specific oligonucleotide for X. fastidiosa strains that cause Pierce's Disease denominated Xf-1 (5'-CGGGGGTGTAGGAGGGGTTGT-3') which was used jointly with the REP-2 primer at the following conditions: 94 ºC/2 min; 35 X (94 ºC/1 min, 62 ºC/1 min; 72 ºC/1 min and 30 s) 72 ºC/10 min. The DNAs isolated from strains of X. fastidiosa from other hosts [almond (Prumus amygdalus), citrus (Citrus spp.), coffee (Coffea arabica), elm (Ulmus americana), mulberry (Morus rubra), oak (Quercus rubra), periwinkle wilt (Catharantus roseus), plums (Prunus salicina) and ragweed (Ambrosia artemisiifolia)] and also from other Gram negative and positive bacteria were submitted to amplification with a pair of primers Xf-1/REP 2 to verify its specificity. A fragment, about 350 bp, was amplified only when the DNA from strains of X. fastidiosa isolated from grapes was employed.

Protection between strains of Passion fruit woodiness virus in sunnhemp

The main objective of the present study was to evaluate the effect of the sunhemp (Crotalaria juncea) host species on the protective ability of two mild strains of Passion fruit woodiness virus (PWV), named F-101 and F-144, which had failed to protect passion flowers (Passiflora edulis f. flavicarpa) in previous experiments. The nucleotide sequences of the capsid protein (CP) gene and the 3'-non-translated region (3'-NTR) of these mild strains and the severe strain of PWV-SP were compared to confirm their relationship. The results of two protective tests with sunhemp plants in the greenhouse and one test under field conditions showed that all plants infected with either mild strain were protected against infection and/or symptom expression of the severe strain of PWV-SP. Evaluation of the relative concentration of the mild strains in sun hemp leaves showed an apparent uniformity in virus distribution in the leaf tissues, different than that which was previously reported for these mild strains in passion flower leaves. These results agree with previous studies that showed the effect of the concentration of the protective strains and the host species in the protection process.

Characterization of Xanthomonas spp. strains by bacteriocins

Twenty-five strains of Xanthomonas axonopodis pv. citri and 14 strains of Xanthomonas spp. were tested for bacteriocin production. X. axonopodis pv. passiflorae strains were sensitive to the bacteriocins produced by the 25 X. axonopodis pv. citri strains evaluated in this study while strains of X. axonopodis pv. manihotis and X. campestris pv. campestris showed variable sensitivity. Only five of the 25 X. axonopodis pv. citri strains were not inhibited by the bacteriocins produced by the two X. axonopodis pv. passiflorae strains. The bacteriocins produced by the Xanthomonas axonopodis pv. citri (FDC-806) and X. axonopodis pv. passiflorae (Mar-2850 A) strains were thermolabile, resistant to lysozyme and sensitive to DNAse. The bacteriocin produced by X. axonopodis pv. passiflorae was resistant to the action of proteinase K, trypsin and RNAse while the bacteriocin produced by X. axonopodis pv. citri was sensitive to these enzymes. The bacteriocins produced by X. axonopodis pv. passiflorae and X. axonopodis pv. citri were called passifloricin and citricin, respectively.

Removal of cyanobacterial toxins by strains of probiotic bacteria

Nedbrytning av blågrönalgtoxiner med hälsobefrämjande mjölksyrebakterier Blomningar av cyanobakterier (blågrönalger) har blivit ett världsomfattande fenomen i eutrofierade vattenmiljöer. Cyanobakterier producerar toxiner, både levergifter och nervgifter, vilka utgör en hälsorisk för människan. Exponeringsrutter omfattar både dricksvatten och förorenade matvaror. Rening av dricksvatten från dessa toxiner är således av hög prioritet. Konventionella vatttenreningsprocesser är inte alltid tillräckligt effektiva mot cyanotoxiner. Därför behövs utveckling av nya effektiva biologiska metoder för vattenrening, vilka kunde komplettera de redan existerande metoderna. FM Sonja Nybom har i sin doktorsavhandling undersökt eliminering av cyanotoxiner från dricksvatten med hjälp av probioter. Probiotiska bakterier, såsom mjölksyrebakterier och bifidobakterier, finns i den naturliga tarmfloran och har även visats ha gynnsamma effekter för människans hälsa. I avhandlingen visades flera olika stammar av probiotiska mjölksyrebakterier och bifidobakterier effektivt eliminera cyanotoxiner, såsom levergiftiga microcystiner, från vatten. Elimineringen undersöktes under olika omständigheter och visades vara beroende av bland annat vattentemperatur, pH, celldensitet och närvaro av kolkälla (glukos) för bakterierna. Metaboliskt aktiva, levande bakterier krävdes för effektiv toxineliminering. En kombination av flera probiotstammar resulterade i effektivare nedbrytning av toxiner i jämförelse med enskilda bakteriestammar. Även reaktionstiden var av betydelse för effektiviteten; efter ett dygns inkubering åstadkoms nästan total nedbrytning. Sammanfattningsvis tyder resultaten på att metoder utnyttjande dessa hälsobefrämjande probiotiska bakterier kunde utvecklas till att användas vid rening av dricksvatten från cyanotoxiner samt användas som en personlig skyddsmekanism mot cyanotoxiner i mag-tarmkanalen.

Water quality indicators for environmental and resistance profile of Escherichia coli strains isolated in Rio Cascavel, Paraná, Brazil

In this article it was evaluated the quality of water in the Cascavel river, in the city of Cascavel - Paraná using microbiological indicators, physical and chemical pollution and susceptibility / resistance in strains of Escherichia coli isolated antimicrobial trade. The water sampling was conducted between 2010-July and 2011-June at three points: a) near the source, b) urban area, c) rural area. The samples were analyzed for physical, chemical and microbiological variables: temperature, pH, color, turbidity, electrical conductivity, total nitrogen and total phosphorus, total coliforms (CT), fecal coliform (CTe) and Escherichia coli. Tests were also performed to nine antimicrobial commercial resistances. The variables studied indicated that the Cascavel river water was presented at disagreement with the resolution 357/2005 CONAMA (class I), ranking in the index as regular water quality. The physical, chemical and rainfall did not affect the growth of CT and CTe, with higher counts of E. coli in the urban area. The greatest resistance profiles of the strains of E. coli isolated from Cascavel river water was found in section 2, the urban area as a probable consequence of human influence on water quality.

Identification and characterisation of a novel adhesin of Streptococcus suis and its use as a target of adhesion inhibition and bacterial detection

Streptococcus suis is an important pig pathogen but it is also zoonotic, i.e. capable of causing diseases in humans. Human S. suis infections are quite uncommon but potentially life-threatening and the pathogen is an emerging public health concern. This Gram-positive bacterium possesses a galabiose-specific (Galalpha1−4Gal) adhesion activity, which has been studied for over 20 years. P-fimbriated Escherichia coli−bacteria also possess a similar adhesin activity targeting the same disaccharide. The galabiose-specific adhesin of S. suis was identified by an affinity proteomics method. No function of the protein identified was formerly known and it was designated streptococcal adhesin P (SadP). The peptide sequence of SadP contains an LPXTG-motif and the protein was proven to be cell wall−anchored. SadP may be multimeric since in SDS-PAGE gel it formed a protein ladder starting from about 200 kDa. The identification was confirmed by producing knockout strains lacking functional adhesin, which had lost their ability to bind to galabiose. The adhesin gene was cloned in a bacterial expression host and properties of the recombinant adhesin were studied. The galabiose-binding properties of the recombinant protein were found to be consistent with previous results obtained studying whole bacterial cells. A live-bacteria application of surface plasmon resonance was set up, and various carbohydrate inhibitors of the galabiose-specific adhesins were studied with this assay. The potencies of the inhibitors were highly dependent on multivalency. Compared with P-fimbriated E. coli, lower concentrations of galabiose derivatives were needed to inhibit the adhesion of S. suis. Multivalent inhibitors of S. suis adhesion were found to be effective at low nanomolar concentrations. To specifically detect galabiose adhesin−expressing S. suis bacteria, a technique utilising magnetic glycoparticles and an ATP bioluminescence bacterial detection system was also developed. The identification and characterisation of the SadP adhesin give valuable information on the adhesion mechanisms of S. suis, and the results of this study may be helpful for the development of novel inhibitors and specific detection methods of this pathogen.

Effect of multidrug resistance modulators on the activity of ivermectin and moxidectin against selected strains of Haemonchus contortus infective larvae

Nematode parasites have shown resistance to the anthelmintics, ivermectin and moxidectin, and there is evidence that the over-expression of parasite P-glycoprotein (P-gp) may account, at least in part, for resistance to ivermectin. The objective of this study was to evaluate whether the multidrug resistance (MDR) modulators, verapamil, CL 347.099 (an analog of verapamil) and cyclosporin A, would enhance the efficacy of ivermectin and moxidectin against selected strains of Haemonchus contortus using an in vitro larval migration assay. The modulators had no effects on the number of migrating larvae when used alone. Ivermectin and moxidectin showed a significant (P<0.05) increase in its efficacy by 52.8 and 58.5% respectively, when used in association with verapamil against a moxidectin-selected strain. CL 347,099 also increased significantly (P<0.05) the ivermectin and moxidectin efficacy by 24.2 and 40.0% respectively, against an ivermectin-selected strain and by 40.0 and 75.6% respectively, against an moxidectin-selected strain. At the concentrations tested cyclosporin A showed a variable effect on increasing the efficacy of the anthelmintics against the susceptible and resistant strains.

Occurrence of F42 colonization factor in Escherichia coli strains isolated from piglets with diarrhea

The objective of this study was to determine the presence of the colonization factor F42 in 168 strains of Escherichia coli isolated from diarrheic stools of newborn piglets. The presence of F42 in 12 (7.1%) strains was detected with the agglutination test. Through the Polymerase Chain Reaction (PCR) of F42 positive strains, gene encoding enterotoxins (ST-I, ST-II, LT-I and LT-II) were detected. The finding of ST-I/ST-II genes in 50% of the strains, ST-I (16%) and ST-II (25%) indicates a strong association of FC F42 with heat-stable enterotoxins (91%). In contrast, the thermolabile enterotoxin (LT-I and LT-II) genes were not detected. Serogroups of F42 positive strains were determined, serogroup O8 being the most prevalent (41,7%). Other serogroups, as there are O9, O11, O18, O32, O35, O98 and O101, were also identified. Thus, FC F42 was confirmed as an additional factor of virulence in the pathogenesis of porcine colibacillosis.

Field evaluation of safety during gestation and horizontal spread of a recombinant differential bovine herpesvirus 1 (BoHV-1) vaccine

Bovine herpesvirus type 1 (BoHV-1) is recognized as a major cause of respiratory, reproductive disease and abortion in cattle. Vaccination is widely applied to minimize losses induced by BoHV-1 infections; however, vaccination of dams during pregnancy with modified live virus (MLV) vaccines has been occasionally associated to abortions. We have previously reported the development of a BoHV-1 recombinant virus, constructed with basis on a Brazilian BoHV-1 (Franco et al. 2002a) from which the gene coding for glycoprotein E (gE) was deleted (gE-) by genetic manipulation. Such recombinant has been previously evaluated in its potential as a differential vaccine (gE- vaccine) that allows differentiation between vaccinated and infected animals. Here, in the first part of the present study, the safety of the gE- vaccine during pregnancy was evaluated by the intramuscular inoculation of 10(7.4) tissue culture 50 % infective doses (TCID50) of the virus into 22 pregnant dams (14 BoHV-1 seronegative; 8 seropositive), at different stages of gestation. Other 15 pregnant dams were kept as non-vaccinated controls. No abortions, stillbirths or fetal abnormalities were seen after vaccination. Seroconversion was observed in both groups of previously seronegative vaccinated animals. In the second part of the study, the potential of the gE- vaccine virus to spread among beef cattle under field conditions was examined. Four heifers were inoculated intranasally with a larger amount (10(7,6) TCID50) of the gE- vaccine (to increase chances of transmission) and mixed with other sixteen animals at the same age and body condition, in the same grazing area, at a population density equal to the average cattle farming density within the region (one cattle head per 10,000 m²), for 180 days. All animals were monitored daily for clinical signs. Serum samples were collected on days 0, 30, 60 and 180 post-vaccination. Seroconversion was observed only in vaccinated heifers. These results indicate that, under the conditions of the present study, the gE- vaccine virus did not cause any noticeable harmful effect on pregnant dams and on its offspring and did not spread horizontally among cattle.

Typing of avian pathogenic Escherichia coli strains by REP-PCR

In the present study the repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) technique was used to establish the clonal variability of 49 avian Escherichia coli (APEC) strains isolated from different outbreak cases of septicemia (n=24), swollen head syndrome (n=14) and omphalitis (n=11). Thirty commensal strains isolated from poultry with no signs of these illnesses were used as control strains. The purified DNA of these strains produced electrophoretic profiles ranging from 0 to 15 bands with molecular sizes varying from 100 bp to 6.1 kb, allowing the grouping of the 79 strains into a dendrogram containing 49 REP-types. Although REP-PCR showed good discriminating power it was not able to group the strains either into specific pathogenic classes or to differentiate between pathogenic and non-pathogenic strains. On the contrary, we recently demonstrated that other techniques such as ERIC-PCR and isoenzyme profiles are appropriate to discriminate between commensal and APEC strains and also to group these strains into specific pathogenic classes. In conclusion, REP-PCR seems to be a technique neither efficient nor universal for APEC strains discrimination. However, the population clonal structure obtained with the use of REP-PCR must not be ignored particularly if one takes into account that the APEC pathogenic mechanisms are not completely understood yet.

Clonal study of avian Escherichia coli strains by fliC conserved-DNA-sequence regions analysis

The clonal relationship among avian Escherichia coli strains and their genetic proximity with human pathogenic E. coli, Salmonela enterica, Yersinia enterocolitica and Proteus mirabilis, was determined by the DNA sequencing of the conserved 5' and 3'regions fliC gene (flagellin encoded gene). Among 30 commensal avian E. coli strains and 49 pathogenic avian E. coli strains (APEC), 24 commensal and 39 APEC strains harbored fliC gene with fragments size varying from 670bp to 1,900bp. The comparative analysis of these regions allowed the construction of a dendrogram of similarity possessing two main clusters: one compounded mainly by APEC strains and by H-antigens from human E. coli, and another one compounded by commensal avian E. coli strains, S. enterica, and by other H-antigens from human E. coli. Overall, this work demonstrated that fliC conserved regions may be associated with pathogenic clones of APEC strains, and also shows a great similarity among APEC and H-antigens of E. coli strains isolated from humans. These data, can add evidence that APEC strains can exhibit a zoonotic risk.