Development of a method for the genetic identification of mussel species belonging to Mytilus, Perna, Aulacomya, and other genera

Legislation regarding the labeling of processed products is an important issue in the protection of consumer rights. This labeling is especially important in products that cannot be identified on the basis of their morphological characters, because these are removed from the animal in the transformation process. The goal of this study was the identification of mussel species using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Forensically Informative Nucleotide Sequencing (FINS) methodologies. The molecular marker selected was 18S rDNA (nuclear small-subunit rDNA gene), which allows identification at the genus level and at the species level in some cases. The genera included in this study were Mytilus, Perna, Aulacomya, Semimytilus, Brachidontes, Choromytilus, and Perumytilus. Different markers were used for genetic identification at the species level. To identify the species included in the genus Perna and Choromytilus, a fragment of ITS 1 (Internal Transcribed Spacer 1) was amplified by multiplex PCR and digested with restrictases. The species of Mytilus were identified by length polymorphism and RFLP of the polyphenolic adhesive protein gene. This methodology was validated with products manufactured in the authors' pilot plant and applied to commercial samples. Therefore, this sequential method can be completely or partially used to determine the mussel genus or species present in any food product.     

Resistance monitoring of Heliothis virescens to pyramided cotton varieties with a hydrateable, artificial cotton leaf bioassay

Proof of concept was demonstrated for a practical, off-the-shelf bioassay to monitor for tobacco budworm resistance to pyramided Bt cotton using plant filtrates. The bioassay was based on a previously described feeding disruption test using hydrateable artificial diet containing a blue indicator dye, a diagnostic dose of insecticide and novel assay architecture. Using neonate larvae from a Bt-susceptible, laboratory reared tobacco budworm strain, a diagnostic dose for Bollgard II and WideStrike cotton was obtained that limited neonate blue fecal production to 0-2 pellets in 24 h (Bt-resistant larvae produced >2 fecal pellets). The bioassay was tested with three different field populations of tobacco budworm collected from tobacco in central North Carolina (USA) and shown to accurately diagnose susceptibility to Bt. The diagnostic doses were also successfully evaluated with two Bt-resistant, laboratory reared tobacco budworm strains. Shelf life studies showed the assay could be stored for at least 6 months at room temperature (longer storage times were not studied). The application of the bioassay as an easy to use monitoring tool is discussed.     

Biodegradability of Disinfectants in Surface Waters from Buenos Aires: Isolation of an Indigenous Strain Able to Degrade and Detoxify Benzalkonium Chloride

Biodegradability of chlorhexidine (CH), triclosan (TC), and benzalkonium chloride (CBA) has been tested in 18 surface water sampling points in the urban area of Buenos Aires. Sampling points were located in both the Reconquista and the Matanza-Riachuelo basins as well as in the La Plata River. High tolerance to the three disinfectants was found and indigenous strains capable of degrading CBA and TC were isolated. Neither tolerance nor biodegradation were correlated with sewage pollution. A strain that degrades CBA was identified as belonging to the genus Pseudomonas using the API20NE system and 16SRNA sequencing. In batch assays, the strain was capable of degrading 100, 200, and up to 500 mg L^?1 of CBA in 10, 25, and 46 h respectively with specific growth rates (μ) of 0.56, 0.30, and 0.14 h^?1. The efficiency of the process was between 99.5–98.0% in terms of compound removal and between 93.8–89.1% in terms of chemical oxygen demand (COD). The detoxification of the compound as a result of the biodegradation was assessed using Pseudokirchneriella subcapitata, Vibrio fischeri, and Lactuca sativa as test organisms.     

Induction of an antioxidant enzyme system and other oxidative stress markers associated with compatible and incompatible interactions between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp.ciceris

To ascertain if active oxygen species play a role in fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris, the degree of lipid peroxidation (malondialdehyde formation) and the activity levels of diamine oxidase (DAO), an apoplastic H₂O₂-forming oxidase, and several antioxidant enzymes, namely ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol-dependent peroxidase (GPX) and superoxide dismutase (SOD), were determined spectrophotometrically in roots and stems of ‘WR315’ (resistant) and ‘JG62’ (susceptible) chickpea cultivars inoculated with the highly virulent race 5 of the pathogen. Moreover, APX, CAT, GPX and SOD were also analysed in roots and stems by gel electrophoresis and activity staining; and the protein levels of APX and SOD in roots were determined by Western blotting. In roots, infection by the pathogen increased lipid peroxidation and CAT and SOD activities, although such responses occurred earlier in the incompatible compared with the compatible interactions. APX, GPX and GR activities were also increased in infected roots, but only in the compatible interaction. In stems, infection by the pathogen increased lipid peroxidation and APX, CAT, SOD and GPX activities only in the compatible interaction, and DAO activity only in the incompatible one. In general, electrophoregrams agreed with the activity levels determined spectrophotometrically and did not reveal any differences in isoenzyme patterns between cultivars or between infected and non-infected plants. Further, Western blots revealed an increase in the root protein levels of APX in the compatible interaction and in those of SOD in both compatible and incompatible interactions. In conclusion, whereas enhanced DAO activity in stems, and earlier increases in lipid peroxidation and CAT and SOD activities in roots, can be associated with resistance to fusarium wilt in chickpea, the induction of the latter three parameters in roots and stems along with that of APX, GR (only in roots) and GPX (only in stems) activities are rather more associated with the establishment of the compatible interaction.     

Evaluation of single and comparative intradermal tuberculin tests for tuberculosis eradication in caprine flocks in Castilla y León (Spain)

Goats can act as reservoirs for tuberculosis (TB) infection. The main etiological agents of TB in goats are Mycobacterium caprae and Mycobacterium bovis and they infect also a wide range of domestic and wild animals and humans. Control programmes based mainly on the application of single and comparative intradermal tuberculin (SIT and SCIT respectively) tests are being implemented in certain regions of Spain with a high density of caprine flocks as Castilla y León, including goats with epidemiological relationship with cattle. The aim of this study was to evaluate the performance of the intradermal tests in naturally TB-infected caprine flocks from this region. The study was performed using data from 17,450 goats in 54 different flocks that were classified as TB-infected in the control programmes executed in 2010 and 2011. Data from 1237 goats from 7 dairy flocks depopulated after the first intradermal testing were used to estimate the sensitivity (Se) using bacteriology as the gold-standard. Overall Se of the SIT test using the severe interpretation was 43.9% (CI 95%, 40.4–47.4) and decreased to 38.8% (CI 95%, 35.5–42.3) using the standard interpretation. Overall Se of the SCIT test ranged between 21.3% (CI 95%, 17.6–25.4) and 7% (CI 95%, 4.9–9.8) depending of the interpretation criteria. A significant weak positive correlation was found between age and skin fold thickness (Spearman’s test p < 0.05). Results from this study yielded, in general, low Se values probably due the systematic detection and slaughter of reactors as a consequence of the eradication programme in previous years or the presence of factors that may interfere in the diagnosis. Therefore, these results suggest the necessity of including ancillary diagnostic tools and/or strict interpretation criteria to maximize detection of positive animals in infected settings.     

Mutants in the lipopolysaccharide of Brucella ovis are attenuated and protect against B. ovis infection in mice

Brucella spp. are Gram-negative bacteria that behave as facultative intracellular parasites of a variety of mammals. This genus includes smooth (S) and rough (R) species that carry S and R lipopolysaccharides (LPS), respectively. S-LPS is a virulence factor, and mutants affected in the S-LPS O-polysaccharide (R mutants), core oligosaccharide or both show attenuation. However, B. ovis is naturally R and is virulent in sheep. We studied the role of B. ovis LPS in virulence by mutating the orthologues of wadA, wadB and wadC, three genes known to encode LPS core glycosyltransferases in S brucellae. When mapped with antibodies to outer membrane proteins (Omps) and R-LPS, wadB and wadC mutants displayed defects in LPS structure and outer membrane topology but inactivation of wadA had little or no effect. Consistent with these observations, the wadB and wadC but not the wadA mutants were attenuated in mice. When tested as vaccines, the wadB and wadC mutants protected mice against B. ovis challenge. The results demonstrate that the LPS core is a structure essential for survival in vivo not only of S brucellae but also of a naturally R Brucella pathogenic species, and they confirm our previous hypothesis that the Brucella LPS core is a target for vaccine development. Since vaccine B. melitensis Rev 1 is S and thus interferes in serological testing for S brucellae, wadB mutant represents a candidate vaccine to be evaluated against B. ovis infection of sheep suitable for areas free of B. melitensis.     

Biometrical, biological, biochemical and molecular characteristics of Meloidogyne incognita isolates and related species

Meloidogyne incognita is one of the most polyphagous species of root-knot nematodes occurring in Brazil and worldwide. Eight M. incognita isolates were studied, representing two enzymatic phenotypes (esterase and malate desydrogenase: I1/N1, I2/N1) and four cryptic Meloidogyne sp.1 (S2/N1) isolates, representing one cytological type (3n?=?40–46). Three M. hispanica isolates (Hi3/N1, 2n?=?32–36) and two of an atypical Meloidogyne sp.2 (S2a/N3, 3n?=?40–44) were included in this study for comparison. All isolates were tested with three M. incognita-specific molecular markers. The primer pairs B06F/R, miF/R and incK14F/R amplified three species-specific fragments of 1,200?bp, 955?bp and 399?bp, respectively for M. incognita and Meloidogyne sp.1 isolates. No amplification occurred in the M. hispanica and Meloidogyne sp.2 isolates, except with primers miF/R (1,650?bp). The genetic variability of the Meloidogyne spp. isolates was evaluated, using RAPD and ISSR markers. The phylogenetic analyses revealed two strongly supported monophyletic clades: clade I, consisting of M. hispanica and the atypical Meloidogyne sp.2 isolates, and clade II, clustering together all M. incognita and the Meloidogyne sp.1 isolates. Considering the biometrical, cytological and molecular approaches, it was possible to conclude that the isolates with three enzymatic phenotypes (I1/N1, I2/N1 and S2/N1) presented the characteristics described for M. incognita. Some correlations were detected between the isozymatic phenotypes and the tree topology (S2a/N3, Hi3/N1, I1/N1, S2/N1), but no strict correlation could be observed for the phenotype I2/N1 and one isolate of S2/N1. Morphologically, the Msp.2 isolates differ from M. incognita and M. hispanica by the female stylet features presenting straight cone tip and round pear shaped knobs, posteriorly sloping. The results of this study suggested that the Msp.2 isolates with phenotypes S2aN3 belong to a new or an unidentified species closely related to M. hispanica.     

Trichoderma as a biological agent of Fusarium oxysporum species complex and Vigna unguiculata growth promoter

European Journal of Plant Pathology - This study aimed to evaluate the potential antagonistic role of Trichoderma in the control of Fusarium isolates belonging to the Fusarium oxysporum species...     

Differences in body temperature, cell viability, and HSP-70 concentrations between Pelibuey and Suffolk sheep under heat stress

Pelibuey and Suffolk sheep were compared as to their capacity to regulate body temperature under environmental hyperthermia by measuring their differences in cellular response to heat stress (HS). In a first experiment, seven Pelibuey and seven Suffolk ewes were kept in a climatic chamber for 6 h daily during 10 days (temperatures within the 18 to 39.5 °C range). As chamber temperature rose, sheep rectal temperature increased in both groups, but to a lesser extent in Pelibuey (0.3 °C) than in Suffolk sheep (0.7 °C) (P?<?0.05). In a second experiment, cellular viability was assessed using cultured blood mononuclear cells from 15 Pelibuey and 15 Suffolk sheep. They were incubated at 37 °C for 24 h (control) or 43 °C for 6 h followed by 18 h at 37 °C (HS). In a third experiment, another blood mononuclear cells culture from eight Pelibuey and eight Suffolk sheep was kept at 37 °C for 15 h; these were subsequently cultured for 6 h at 37 °C (controls) or 43 °C (HS). Next, HSP-70 concentration was determined. HS reduced the percentage of viable cells to a greater extent in Suffolk [37 °C (73.7 %) vs. 43 °C (61.9 %); P?<?0.05] than in Pelibuey sheep [37 °C (74.9 %) vs. 43 °C (66.7 %); P?>?0.05]. HS significantly increased HSP-70 average concentrations for both breeds at 43 °C. A significant effect was observed for the breed by temperature interaction (P?<?0.05) caused by a greater difference between Pelibuey and Suffolk at 43 °C (2.85 vs. 0.53 ng/mL, respectively; P?<?0.05) than at 37 °C (0.05 vs. 0.03 ng/mL, respectively; P?>?0.05). In conclusion, Pelibuey sheep show more effective body temperature regulation under conditions of environmental hyperthermia. Also, cell viability after HS was higher in Pelibuey than in Suffolk, an effect that could be mediated by an HSP-70-related mechanism.