Single-strand Conformation Polymorphism Profiles Research Articles

Application of biological and single-strand conformation polymorphism assays for characterizing potential mild isolates of Citrus tristeza virus for cross protection

Citrus tristeza virus (CTV) by killing millions of citrus cultivars grown on sour orange rootstock worldwide has become one of the most dangerous viral pathogen. Characterization of 12 CTV isolates was analyzed by biological indexing. Infected samples of citrus were collected from citrus growing areas of Pakistan and CTV was detected by symptoms on indicator plants and confirmed by direct tissue blot immunoassay (DTBIA). CTV positive samples were graft inoculated on six biological indicator hosts in the study. A standardized protocol was deployed to study biological characteristics of these isolates. All biological indicators induced mild and from mild to moderate reactions against all of the CTV isolates tested. About two isolates produced stem-pitting symptoms from moderate to severe on Mexican lime. CTV strains were further characterized and confirmed by the analysis of p25 gene of CTV isolates using single-strand conformation polymorphism (SSCP) assay. SSCP analysis revealed that most isolates confined only one predominant sequence variant. SSCP profiles of PCR amplified products from CTV isolates showed bands patterns corresponding to mild and sever strain. Three isolates (4MF, 8KBS and 10GS) from different regions and cultivars were identified as potential source of mild strains for cross protection. These results are the best base for mild strain cross protection (MSCP) in the country.

Bacterial Community Structure and Functional arrA Gene Diversity Associated with Arsenic Reduction and Release in an Industrially Contaminated Soil

ABSTRACTThis study aimed at evaluating potential arsenic (As) mobility in an industrially contaminated soil (64 mg/kg of As) of the Meuse River basin, and at identifying key bacterial groups that drive soil As dynamics. Both speciation and release of As from this soil was followed under anaerobic conditions using a laboratory batch experiment. In the presence of exogenous carbon sources, AsV initially present in the soil matrix and/or adsorbed on synthetic hydrous ferric oxides were solubilized and mainly reduced to AsIII by indigenous soil microflora. After a 1-month incubation period in these biotic conditions, AsIII accounted for 80–85% of the total dissolved As and more than 60% of the solid-phase As. Bacterial community structure (i.e., 16S rDNA-based capillary electrophoresis single-strand conformation polymorphism profiles) changed with incubation time and As amendment. The detection of distantly related arsenate respiratory reductase genes (arrA), as functional markers of AsV respirers, indicates that novel dissimilatory AsV-reducing bacteria may be involved in As biotransformation and mobility in anoxic soils. Since As and iron were concomitantly released, a crucial role of indirect As-mobilizing bacteria on As behavior was also revealed. Our results show that the majority of As within the soil matrix was bioavailable and bioaccessible for heterotrophic AsV reduction to AsIII, which may increase As toxicity and mobility in the contaminated soils.

Effect of salinity and temperature on the bacterial diversity shift of anaerobic batch cultures treating abattoir wastewater

AbstractThe molecular biological analyses allowed to highlight the changes of the microflora of the batch cultures with the increase of the salinity and the temperatures. The single-strand conformation polymorphism (SSCP) patterns of the bacterial diversity at different salt concentrations (0, 20, and 40 g l−1) in mesophilic (37°C) and thermophilic (55°C) conditions showed that the bacterial diversity varies depending on the culture conditions. The obtained SSCP profiles at the different salt concentrations showed that there is a greater diversity in the mesophilic than the thermophilic condition. However, the bacterial diversity richness (1/D) and the species evenness for the mesophilic condition for all the tested salt concentrations showed firstly the maximum diversity and secondly that the species in the sample are quite evenly distributed. The increase of the salt concentration to 20 and 40 g l−1 at thermophilic condition decreased the bacterial diversity due to the selection pressure caused by the e...

Experimental study of the impact of antimicrobial treatments on Campylobacter, Enterococcus and PCR-capillary electrophoresis single-strand conformation polymorphism profiles of the gut microbiota of chickens.

An experiment was conducted to compare the impact of antimicrobial treatments on the susceptibility of Campylobacter, Enterococcus faecium and Enterococcus faecalis, and on the diversity of broiler microbiota. Specific-pathogen-free chickens were first orally inoculated with strains of Campylobacter and Enterococcus faecium. Birds were then orally treated with recommended doses of oxytetracycline, sulfadimethoxine/trimethoprim, amoxicillin or enrofloxacin. Faecal samples were collected before, during and after antimicrobial treatment. The susceptibility of Campylobacter, Enterococcus faecium and Enterococcus faecalis strains isolated on supplemented or non-supplemented media was studied and PCR-capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) profiles of the gut microbiota were analysed. Enrofloxacin-resistant Campylobacter were selected in the enrofloxacin-treated group and showed the Thr86Ile mutation in the gyrA gene. Acquisition of the tetO gene in Campylobacter coli isolates was significantly more frequent in birds given oxytetracycline. No impact of amoxicillin treatment on the susceptibility of Campylobacter could be detected. Ampicillin- and sulfadimethoxine/trimethoprim-resistant Enterococcus faecium were selected in amoxicillin-treated broilers, but no selection of the inoculated vancomycin-resistant Enterococcus faecium could be detected, although it was also resistant to tetracycline and sulfadimethoxine/trimethoprim. PCR-CE-SSCP revealed significant variations in a few peaks in treated birds as compared with non-treated chickens. In conclusion, antimicrobial treatments perturbed chicken gut microbiota, and certain antimicrobial treatments selected or co-selected resistant strains of Campylobacter and Enterococcus.

Discovery of Novel Rickettsiella spp. in Ixodid Ticks from Western Canada

The genomic DNA from four species of ixodid ticks in western Canada was tested for the presence of Rickettsiella by PCR analyses targeting the 16S rRNA gene. Eighty-eight percent of the Ixodes angustus (n = 270), 43% of the I. sculptus (n = 61), and 4% of the I. kingi (n = 93) individuals examined were PCR positive for Rickettsiella, whereas there was no evidence for the presence of Rickettsiella in Dermacentor andersoni (n = 45). Three different single-strand conformation polymorphism profiles of the 16S rRNA gene were detected among amplicons derived from Rickettsiella-positive ticks, each corresponding to a different sequence type. Furthermore, each sequence type was associated with a different tick species. Phylogenetic analyses of sequence data of the 16S rRNA gene and three other genes (rpsA, gidA, and sucB) revealed that all three sequence types were placed in a clade that contained species and pathotypes of the genus Rickettsiella. The bacterium in I. kingi represented the sister taxon to the Rickettsiella in I. sculptus, and both formed a clade with Rickettsiella grylli from crickets (Gryllus bimaculatus) and "R. ixodidis" from I. woodi. In contrast, the Rickettsiella in I. angustus was not a member of this clade but was placed external to the clade comprising the pathotypes of R. popilliae. The results indicate the existence of at least two new species of Rickettsiella: one in I. angustus and another in I. kingi and I. sculptus. However, the Rickettsiella strains in I. kingi and I. sculptus may also represent different species because each had unique sequences for all four genes.

Mutation scanning-based identification of larval and nymphal ticks (Acari: Ixodidae) from Richardson's ground squirrels (Spermophilus richardsonii)

The objective of this study was to identify the tick species parasitizing Richardson's ground squirrels (Spermophilus richardsonii) in southern Saskatchewan (Canada). Morphological examination of the adult ticks revealed the presence of three tick species, Ixodes sculptus, Ixodes kingi and Dermacentor andersoni. However, given the difficulties in identifying some of the larval and nymphal (immature) ticks using this approach, PCR-based single-strand conformation polymorphism (SSCP) and DNA sequence analyses of a portion of the mitochondrial (mt) 16S rRNA gene were used to determine their species identity. The results showed that each tick species had a unique set of SSCP profiles and DNA sequences using this mt marker. The species identity of larval and nymphal ticks was determined based on a comparison of these profiles and sequences with those of morphologically-identified adults. The detection of three tick species, which are known vectors of disease-causing agents, on the same host has important implications for understanding the ecology of vector-borne diseases, and provides an opportunity to examine fundamental questions regarding the structure and composition of the bacterial communities (i.e., both endosymbiotic and pathogenic species) in these ticks. This study shows the utility and benefits of using the present molecular method for the accurate identification of ticks at any stage of development.

Single-Strand Conformation Polymorphism for Molecular Variability Studies of Six Viroid Species

Molecular diversity within six viroid species and different molecular variants, in each species infecting fruit trees was first estimated by the single-strand conformation polymorphism (SSCP) technique and then by direct sequencing analysis. The different variants studied are to three Australian grapevine viroids(AGVd), four citrus dwarfing viroids (CDVd), eleven grapevine yellow speckle viroids type-1 (GYSVd-1), four hop stunt viroids (HSVd), seven peach latent mosaic viroids (PLMVd), and eight pear blister canker viroids (PBCVd). Polyacrylamide gel electrophoresis (PAGE) conditions were compared and optimized to improve the sensitivity of the existing SSCP parameters. The relationships among the various SSCP profiles observed and the variation in nucleotide sequences was studied. The results indicate that the variations of some parameters of electrophoresis for each species allowed higher resolution and hence detection of single nucleotide variations among clones initially clustered into the same group.

Elevated CO2 and drought stress effects on the chemical composition of maize plants, their ruminal fermentation and microbial diversity in vitro

Climate changes are supposed to influence productivity and chemical composition of plants. In the present experiments, it was hypothesised that the incubation of plants exposed to elevated atmospheric carbon dioxide concentrations ([CO2]) and drought stress will result in different ruminal fermentation pattern and microbial diversity compared to unaffected plants. Maize plants were grown, well-watered under ambient (380 ppm CO2, Variant A) and elevated [CO2] (550 ppm CO2, Variant B). Furthermore, each CO2 treatment was also exposed to drought stress (380 ppm and 550 ppm CO2,Variants C and D, respectively), which received only half as much water as the well-watered plants. Plant material from these treatments was incubated in a semi-continuous in vitro fermentation experiment using the rumen simulation technique. Single strand conformation polymorphism (SSCP) analysis was conducted for Bacteria and Archaea specific profiles. The analysis of crude nutrients showed higher contents of fibre fraction in drought stress Variants C and D. Crude protein content was increased by drought stress under ambient but not under elevated [CO2]. Fermentation of drought stress variants resulted in significantly increased pH values, decreased digestibilities of organic matter and increased ammonia–N (NH3–N) concentrations compared with well-watered variants. Additionally, the 550 ppm CO2 Variants B and D showed significantly lower NH3–N concentrations than Variants A and C. The Bacteria- and Archaea-specific SSCP profiles as well as the production rates of short-chain fatty acids and their molar percentages were not affected by treatments. During the first four days of equilibration period, a decrease of molar percentage of acetate and increased molar percentages of propionate were observed for all treatments. These alterations might have been induced by adaptation of the in vitro system to the new substrate. The rumen microflora appeared to be highly adaptive and could cope with altered contents of crude nutrients in plants as induced by elevated [CO2] and drought stress.

Characterization of the microbial diversity in production waters of mesothermic and geothermic Tunisian oilfields

The microbial diversity of production waters of five Tunisian oilfields was investigated using Single Strand Conformation Polymorphism (SSCP) technique followed by cloning-sequencing. Dynamics of bacterial populations in production waters collected from four wellheads were also evaluated. For all production water samples collected, DNA from Archaea and Eucarya was not sufficiently abundant to permit detection rRNA genes from these groups by PCR-SSCP. In contrast, the bacterial rRNA genes were detected in all samples, except for samples from DOULEB12 and RAMOURA wells. SSCP profiles attested that two of the studied geothermic wells (ASHTART47 and ASHTART48) had shown a clear change over time, whereas a stable diversity was found with the mesophilic DOULEB well (DL3). PCR amplification of rRNA genes was unsuccessful with samples from DOULEB (DL12) at all three sampling time. The bacterial diversity present in production waters collected from pipelines of SERCINA and LITAYEM oilfields was high, while production waters collected from wellheads (ASHTART and DOULEB) exhibited lower diversity. The partial study of the biodiversity showed a dominance of uncultured bacteria and Pseudomonas genus (class of the Gammaproteobacteria) in three of the studied oilfields (ASHTART47, ASHTART48 and DOULEB3). However, for LITAYEM oilfield, a significant dominance of 5 phyla (Proteobacteria, Thermotogae, Firmicutes, Synergistetes, Bacteroidetes) was shown. Our study gives a real picture of the microbiology of some Tunisian oilfield production waters and shows that some of the sequenced bacterial clones have a great similarity to previous sequenced clones described from other oilfields all over the world, indicating that these ecosystems harbour specific microbial communities. These findings can be considered as an indirect indication of the indigenous origin of these clones.

Influence of the maize silage to grass silage ratio and feed particle size of rations for ruminants on the community structure of ruminal Firmicutes in vitro

To investigate the effect of the forage source and feed particle size (FPS) in ruminant rations on the composition of the ruminal Firmicutes community in vitro. Three diets, varying in maize silage to grass silage ratio and FPS, were incubated in a rumen simulation system. Microbial samples were taken from the liquid fermenter effluents. Microbial community analysis was performed by 16S rRNA-based techniques. Clostridia-specific single-strand conformation polymorphism profiles revealed changes of the community structure in dependence on both factors tested. The coarse grass silage-containing diets seemed to enhance the occurrence of different Roseburia species. As detected by real-time quantitative PCR, Ruminococcus albus showed a higher abundance with decreasing FPS. A slightly lower proportion of Bacilli was found with increasing grass silage to maize silage ratio by fluorescence in situ hybridization (FISH). In contrast, a slightly higher proportion of bacterial species belonging to the Clostridium-clusters XIV a and b was detected by FISH with increasing grass silage contents in the diet. The ruminal Firmicutes community is affected by the choice of the forage source and FPS. This study supplies fundamental knowledge about the response of ruminal microbial communities to changing diets. Moreover, the data suggest a standardization of grinding of feeds for in vitro studies to facilitate the comparison of results of different laboratories.

Bacterial community structure in experimental methanogenic bioreactors and search for pathogenic clostridia as community members

Microbial conversion of organic waste or harvested plant material into biogas has become an attractive technology for energy production. Biogas is produced in reactors under anaerobic conditions by a consortium of microorganisms which commonly include bacteria of the genus Clostridium. Since the genus Clostridium also harbors some highly pathogenic members in its phylogenetic cluster I, there has been some concern that an unintended growth of such pathogens might occur during the fermentation process. Therefore this study aimed to follow how process parameters affect the diversity of Bacteria in general, and the diversity of Clostridium cluster I members in particular. The development of both communities was followed in model biogas reactors from start-up during stable methanogenic conditions. The biogas reactors were run with either cattle or pig manures as substrates, and both were operated at mesophilic and thermophilic conditions. The structural diversity was analyzed independent of cultivation using a PCR-based detection of 16S rRNA genes and genetic profiling by single-strand conformation polymorphism (SSCP). Genetic profiles indicated that both bacterial and clostridial communities evolved in parallel, and the community structures were highly influenced by both substrate and temperature. Sequence analysis of 16S rRNA genes recovered from prominent bands from SSCP profiles representing Clostridia detected no pathogenic species. Thus, this study gave no indication that pathogenic clostridia would be enriched as dominant community members in biogas reactors fed with manure.

Three genetic grapevine leafroll-associated virus 3 variants identified from South African vineyards show high variability in their 5′UTR

Three genetic variants of grapevine leafroll-associated virus 3 (GLRaV-3) were identified in vineyards of the Western Cape, South Africa. The GLRaV-3 variants were identified by single-strand conformation polymorphism (SSCP) profiles generated from a region amplified in ORF5. ORF5 sequence data confirmed the three genetic variant groups, and a specific SSCP profile was assigned to each variant group. The results of SSCP analysis of this region in ORF5 showed that this method gives a fast and reliable indication of the GLRaV-3 variant status of a plant, which in many instances showed mixed infections. The full genome sequence of one representative of each variant group i.e. isolates 621 (group I), 623 (group II) and PL-20 (group III), was determined by sequencing overlapping cloned fragments of these isolates. The sequences of genomic 5' ends of these isolates were determined by RLM-RACE. Sequence alignment of the 5'UTRs indicated significant sequence and length variation in this region between the three South African variant groups. Alignment of the Hsp70h and CP gene regions of these isolates with those of isolates from elsewhere in the world, followed by phylogenetic analysis, further supported the presence of three variants of GLRaV-3 in South Africa and the presence of two or three additional variant groups elsewhere in the world.

Multiple Infection of Apple Trees by Distinct Strains of ‘Candidatus Phytoplasma mali’ and Its Pathological Relevance

Forty-eight apple trees infected by 'Candidatus Phytoplasma mali' were examined using single-strand conformation polymorphism (SSCP) and sequence analysis of a variable hflB gene fragment and the immunodominant membrane protein-encoding imp gene. SSCP analysis of polymerase chain reaction-amplified hflB gene fragments revealed diverse profiles, differing in number and position of the bands. The 'Ca. P. mali' content of a single infected apple tree was termed an accession. Cloning of fragments from accessions that yielded fewer bands resulted in clone populations showing uniform or moderately polymorphic SSCP patterns and largely homogenous nucleotide sequences. In contrast, inserts from accessions yielding more bands were heterogeneous and formed two to four distinct groups of profiles. DNA fragments from such accessions were diverse and clustered distantly when subjected to phylogenetic analysis, mostly as two homogenous groups plus one or a few other sequences. Similar results were obtained upon imp gene examination. The collective data indicate that accessions exhibiting more complex patterns were composed of two or three distinct 'Ca. P. mali' strains. There is evidence that multiple infections are of pathological relevance due to strain interactions leading to shifts in the populations. In triply infected trees of one accession, no specific symptoms were induced by the presence of two of the strains. The rare appearance of pronounced symptoms was associated with a separate strain that possessed a unique SSCP profile and a unique hflB sequence. The two mild strains from this apple accession also induced only mild symptoms on periwinkle and tobacco and occurred specifically in one of these plants.

Performance of a sulfidogenic bioreactor and bacterial community shifts under different alkalinity levels

The performance of a sulfidogenic bioreactor and the response of bacterial populations to influent alkalinity changes were investigated. The bioreactor reached 40% of sulfate removal efficiency (SRE) with 0 mg l(-1) of alkalinity, and single-stranded conformation polymorphism profiles showed that some members of Bacteroides, Dysgonomonas, Sporobacter, Quinella, and Citrobacter became dominant populations. 16S rRNA gene library analysis indicated that the Actinobacteria group increased from 0% in seed to 23% in sludge. An increase in alkalinity to 1300 mg l(-1) led to a rapid increase of SRE to 65% and changes in the bacterial community. Sequences representing Dysgonomonas, Raoultella, Kluyvera, and Phascolarctobacterium were now found. When alkalinity was deceased to 0 mg l(-1), SRE dropped and the bands representing Raoultella, Kluyvera, and Phascolarctobacterium disappeared, while bands representing Clostridium appeared. A second cycle of low/high alkalinity did not result in obvious changes to the bacterial community. These results indicate that the sulfidogenic bioreactor favored higher influent alkalinity and that the different functional microbial populations responded well to the alkalinity changes.

Stability of Citrus tristeza virus protective isolates in field conditions

The objective of this work was to monitor the maintenance of Citrus tristeza virus (CTV) protective isolates stability in selected clones of 'Pêra' sweet orange (Citrus sinensis), preimmunized or naturally infected by the virus, after successive clonal propagations. The work was carried out in field conditions in the north of Paraná State, Brazil. Coat protein gene (CPG) analysis of 33 isolates collected from 16 clones of 'Pêra' sweet orange was performed using single strand conformational polymorphism (SSCP). Initially, the isolates were characterized by symptoms of stem pitting observed in clones. Then viral genome was extracted and used as template for the amplification of CPG by reverse transcription polimerase chain reaction (RTPCR). RTPCR products electrophoretic profiles were analyzed using the Jaccard coefficient and the UPGMA method. The majority of the clones had weak to moderate stem pitting symptoms and its CTV isolates showed alterations in the SSCP profiles. However, the stability of the protective complex has been maintained, except for isolates from two analised clones. Low genetic variability was observed within the isolates during the studied years.

Effect of the corn silage to grass silage ratio and feed particle size of diets for ruminants on the ruminal Bacteroides-Prevotella community in vitro

This study examined whether different corn silage to grass silage ratios in ruminant rations and different grinding levels of the feed affect the composition of the ruminal Bacteroides-Prevotella community in vitro. Three diets, composed of 10% soybean meal as well as of different corn silage and grass silage proportions, were ground through 1 mm or 4 mm screened sieves and incubated in a semi-continuous rumen simulation system. On day 14 of the incubation microbes were harvested by centrifugation from the liquid effluent of fermenter vessels. Microbial DNA was extracted for single strand conformation polymorphism (SSCP) analysis of 16S rRNA genes followed by sequencing of single SSCP bands. Fluorescence in situ hybridization (FISH) and real-time quantitative (q) PCR were used to quantify differences in the relative abundance of Bacteroides-Prevotella and Prevotella bryantii. SSCP profiles revealed a significant influence of the forage source as well as of the feed particle size on the community structure of the Bacteroides-Prevotella group. Different, phylogenetically distinct, so far uncultured Prevotella species were detected by sequence analysis of several treatment-dependent occurring SSCP bands indicating different nutritional requirements of these organisms for growth. No quantitative differences in the occurrence of Bacteroides-Prevotella-related species were detected between diets by FISH with probe BAC303. However, real-time qPCR data revealed a higher abundance of P. bryantii with increasing grass silage to corn silage ratio, thus again indicating changes within the community composition of the Bacteroides-Prevotella group. As P. bryantii possesses high proteolytic activity its higher abundance may have been caused by the higher contents of crude protein in the grass silage containing diets. To conclude, results of this study show an influence of the forage source on the ruminal community of Bacteroides-Prevotella. Furthermore, they suggest an effect of the feed particle size on this bacterial group.

Is microbial diversity an asset for inhibitingListeria monocytogenesin raw milk cheeses?

This study aimed at determining if microbial diversity can be an asset to guarantee the microbial safety of raw milk cheeses. Our results show that microbial consortia from the surface of raw milk cheeses can self-protect against Listeria monocytogenes. Indeed, 10 complex microbial consortia among 34 tested from the surfaces of raw milk Saint-Nectaire cheeses were particularly effective for reducing the growth of L. monocytogenes on cheese surfaces in comparison of a commercial ripening culture, despite the high pH values on the surfaces. One of these consortia (TR15) was selected and propagated on cheese surfaces to create a collection of strains belonging to lactic acid bacteria, Gram-positive and catalase-positive bacteria, Gram-negative bacteria and yeasts. On the surfaces of uncooked cheeses, defined consortia consisting of combinations of several isolates from this collection displayed weaker antagonist activity against L. monocytogenes than the complex consortium TR15. The results from plate counting and analysis by single strand conformation polymorphism (SSCP) converged to show that microbial dynamics in cheeses TR15 differed from that of the defined consortia. TR15 cheeses had the highest levels of cultivable lactobacilli and leuconostocs. Their SSCP profiles were the richest in peaks and were characterised by the presence of Marinilactibacillus psychrotolerans, Carnobacterium mobile, Arthrobacter nicotianae or A. arilaitensis, Arthrobacter ardleyensis or A. bergerei and Brachybactrerium sp. Further investigation will be necessary to gain a better understanding of the microbial interactions involved in inhibiting L. monocytogenes.

Search of environmental descriptors to explain the variability of the bacterial diversity from maize rhizospheres across a regional scale

The regional scale variability of the bacterial community inhabiting the rhizosphere was studied with soil collected from maize fields located in the Santo Domingo Valley (SDV; Baja California Sur, Mexico), a semi-arid agricultural ecosystem of approximately 200 km 2. The bacterial community structure was visualized by single-strand conformation polymorphism (SSCP) profiles of PCR-amplified partial 16S rRNA genes of directly extracted rhizosphere soil DNA. SSCP profiles of different SDV sites and an external field site in Germany were evaluated for their similarities and the contributing bacteria were characterized by DNA sequence analyses. SSCP profiles from each site were significantly different from the others, as revealed by permutation of pairwise similarities ( P < 0.05). In comparison to the German site, SSCP profiles from SDV were more similar to each other despite contrasting soil salinity levels. Correspondence analysis revealed that among SDV sites, salinity levels, soil organic carbon and calcium (Ca 2+) were most influential on the bacterial community structure. Depending on the phylogenetic group analyzed ( Bacteria, Alphaproteobacteria, Pseudomonas), the importance of these soil variables varied. Interestingly, the East–West direction also revealed an effect, suggesting that future explorations of bacterial diversity patterns should also consider landscape topography in search of explaining patterns of bacterial diversity in soils.

Species identification using a small nuclear gene fragment: application to sympatric wild carnivores from South-western Europe

Species identification is essential for non-invasive studies of elusive and rare animals, and for detecting illegal harvest or trade of wildlife species. However, most molecular tests identify only a limited number of species or require multiple laboratory steps to distinguish many taxa. Additionally, most protocols use mitochondrial DNA being, therefore, especially prone to problems such as nuclear insert copies, high intraspecific diversity or heteroplasmy. Here, we developed a molecular test based on the polymorphisms detected on a small nuclear gene fragment (221 bp of the IRBP -Interphotoreceptor Retinoid-Binding Protein- exon 1). This fragment revealed 51 variable sites (including 12 non-synonymous and 19 species-specific sites), which enabled the successful distinction of all 16 carnivore species native to South-western Europe. A SSCP (Single-Strand Conformational Polymorphism) gel electrophoresis technique was also optimized to allow the simple and inexpensive application of this molecular test. Sequences and SSCP profiles were consistent in identifying a total of 387 samples, including faeces (172) and hairs (17) collected non-invasively in the field. Due to its low cost, simplicity, and wide range of identifiable species, this test shows great promise to facilitate studies in molecular ecology, conservation genetics, and forensic analysis, as well as DNA bar-coding projects.

Do milking practices influence the bacterial diversity of raw milk?

The link between milk production practices and bacterial diversity of 67 raw milks from dairy farms in the Savoie and Haute-Savoie regions of France was studied by Single Strand Conformation Polymorphism (SSCP) analysis. The milking practices and the cleanliness of different parts of the cow housing were evaluated. The SSCP bacterial profiles allow to classify the 67 milks into three groups: group A was characterised by a majority of Gram-positive non-lactic acid bacteria (Corynebacterineae and Micrococcaceae) and a high level of hygiene in milking practices. The SSCP profiles of groups B and C were close but different from those of group A: they were both dominated by lactic acid bacteria and by a less intensive hygiene practices. The group B milks were characterised by the dominance of Gram-negative bacteria and Lactococcus lactis species while those of group C were dominated by Brevibacterium linens and Leuconostoc mesenteroides. The variation of balance between bacterial populations can be associated with differences in hygienic milking production practices.