Detection and quantification of Anaplasma marginale DNA in blood samples of cattle by real-time PCR

A TaqMan-based real-time PCR assay was developed for the diagnosis of Anaplasma marginale infection of cattle. The established assay was proven to be highly specific, since no cross-reactions were observed with other Anaplasma species of ruminants, including the closely related Anaplasma centrale, or other haemoparasites of ruminants (Anaplasma bovis, Anaplasma ovis, Anaplasma phagocytophilum, Babesia bovis, Babesia bigemina, Theileria annulata and Theileria buffeli). The detection limit was equal to that of nested (n)PCR (10(1) copies of standard DNA and 3 x 10(1) infected erythrocytes ml(-1) of blood). The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. Fifty-four blood samples of ruminants (cattle, n = 51; sheep, n = 2; goats, n = 1), that had been tested previously by reverse line blot (RLB) hybridisation, were subjected to an nPCR assay and the newly established real-time PCR assay. By using real-time PCR, A. marginale DNA was detected in 39/51 bovine samples, with DNA titres ranging from 3.60 x 10(3) to 5.70 x 10(8) copies ml(-1) of blood, whereas sheep and goat samples tested negative. The concordance with nPCR was 100%, whereas a unique sample that had tested negative by RLB gave positive results by nPCR and real-time PCR. The established assay could overcome the limitations of existing diagnostic methods, allowing for simultaneous detection and quantification of the A. marginale DNA in bovine blood, that is essential to support the clinical diagnosis, to assess the carrier status of the animals and to evaluate the efficacy of vaccines and antirickettsial drugs.     

Relationship between milk fatty acid composition and dietary roughage source in dairy cows

According to a 2 × 2 crossover design, 14 Holstein dairy cows were fed two isoenergetic diets based on either grass hay (GH) or maize silage (MS). Milk samples were collected during the third week of each period, and fatty acid (FA) profiles were analyzed using gas chromatography. The data obtained were subjected to ANOVA. Dietary treatment had no effect on either dry matter intake or milk yield. Milk from animals fed the GH-diet contained lower concentrations of saturated FAs (61.9 vs. 63.4% of total FAs; P < 0.05) and higher levels of polyunsaturated FAs (PUFAs) (6.1 vs. 5.8; P < 0.01). Feeding additional hay also increased conjugated linoleic acid and n-3 FA levels and decreased C16:0 levels. Increases in both PUFAs and n-3 FAs resulted in lower (P < 0.01) atherogenic and thrombogenic indices in milk from animals fed the GH diet compared with those fed the MS diet. A complete substitution of GH for MS appeared to improve milk FA profiles, even using different types of concentrates to provide a balanced diet.

     

Isolation and determination of alpha-dicarbonyl compounds by RP-HPLC-DAD in green and roasted coffee

Glyoxal, methylglyoxal, and diacetyl formed as Maillard reaction products in heat-treated food were determined in coffee extracts (coffee brews) obtained from green beans and beans with different degrees of roast. The compounds have been reported to be mutagenic in vitro and genotoxic in experimental animals in a number of papers. More recently, alpha-dicarbonyl compounds have been implicated in the glycation process. Our data show that small amounts of glyoxal and methylglyoxal occur naturally in green coffee beans. Their concentrations increase in the early phases of the roasting process and then decline. Conversely, diacetyl is not found in green beans and forms later in the roasting process. Therefore, light and medium roasted coffees had the highest glyoxal and methylglyoxal content, whereas dark roasted coffee contained smaller amounts of glyoxal, methylglyoxal, and diacetyl. For the determination of coffee alpha-dicarbonyl compounds, a reversed-phase high performance liquid chromatography with a diode array detector (RP-HPLC-DAD) method was devised that involved the elimination of interfering compounds, such as chlorogenic acids, by solid phase extraction (SPE) and their derivatization with 1,2-diaminobenzene to give quinoxaline derivatives. Checks of SPE and derivatization conditions to verify recovery and yield, respectively, resulted in rates of 100%. The results of the validation procedure showed that the proposed method is selective, precise, accurate, and sensitive.     

Timing and order of different insecticide classes drive control of Drosophila suzukii; a modeling approach

The spotted-wing drosophila, Drosophila suzukii Matsumura, is an invasive pest causing significant damage to soft skinned fruits. Control of D. suzukii is critical since there is no tolerance for infested fruit in the market. While most insecticides control one or more D. suzukii life-stages (e.g., egg, larvae, and adult), the impact of insecticides that are toxic to immature stages  is unclear on the subsequent generation of a field population. Insecticides were applied at field recommended rates on cherries and blueberries in the laboratory to determine immature D. suzukii mortality. Spinetoram, cyantraniliprole, malathion, methomyl, spinosad, and phosmet resulted in relatively high mortality of all immature life stages. Zeta-cypermethrin, cyclaniliprole, and fenpropathrin resulted in lower mortality of egg and all larval instars. Malathion was also applied to lowbush blueberries with different fruit sizes (small, medium, and large) in the laboratory and there was no statistical difference in mortality rates depending on fruit sizes. Mortality data from the laboratory experiments were used to parameterize a refined D. suzukii population model. The model revealed that the timing and order of different insecticide classes are important to control D. suzukii population. Model runs that included early applications of more effective insecticides resulted in high immature mortality and greater reduction of D. suzukii populations compared to treatments applied later.

     

Gamna‐Gandy bodies: a case of mistaken identity in the spleen of a cat

A 4‐year‐old, female spayed Domestic Shorthair cat was presented for a 2‐day history of lethargy, anorexia, and vomiting. Diagnostic assessments revealed a severe, regenerative anemia and the presence of a solitary, 3 × 4 cm splenic mass. Cytologic examination of fine‐needle aspirates of the splenic mass showed sheets of pleomorphic histiocytic cells with numerous intracellular and extracellular, negatively staining to deeply basophilic, branching structures. These structures were typically of thin uniform thickness with random angled branching that formed geometrical shapes and fragments, occasionally with sharpened ends and a crystalline appearance. Septal‐like divisions were irregularly spaced along some of the linear branches. The cytology was interpreted as a probable histiocytic inflammatory response to the unidentified foreign material with histiocytic sarcoma as a differential diagnosis. Histopathologic evaluation of the splenic mass resulted in a diagnosis of histiocytic sarcoma. The branching material stained positively for both iron and calcium, indicating that the material was compatible with Gamna‐Gandy bodies present secondary to tumor‐associated hemorrhage and the formation of calcium–iron complexes.     

Antiadhesion and antibiofilm activities of high molecular weight coffee components against Streptococcus mutans

In previous studies we demonstrated that green and roasted coffee contains low molecular weight (LMW) compounds capable of inhibiting the ability of Streptococcus mutans, the major causative agent of human dental caries, to adhere to hydroxyapatite (HA) beads. This study addressed the ability of the whole high molecular weight coffee fraction (cHMW) and of its melanoidin and non-melanoidin components (GFC1-5), applied at concentrations that occur in coffee beverages, to (i) inhibit S. mutans growth; (ii) affect S. mutans sucrose-dependent adhesion to and detachment from saliva-coated HA beads (sHA); and (iii) inhibit biofilm development on microtiter plates. The results indicated that only cHMW is endowed with antimicrobial activity. The cHMW fraction and each of the five GFC components inhibited S. mutans adhesion, the strongest effect being exerted by cHMW (91%) and GFC1 (88%). S. mutans detachment from sHA was four times greater (～20%) with cHMW and the GFC1 and GFC4 melanoidins than with controls. Finally, biofilm production by S. mutans was completely abolished by cHMW and was reduced by 20% by the melanoidin components GFC2 and GFC4 and by the non-melanoidin component GFC5 compared with controls. Altogether these findings show that coffee beverage contains both LMW compounds and HMW melanoidin and non-melanoidin components with a strong ability to interfere in vitro with the S. mutans traits relevant for cariogenesis.     

Nitrogen metabolism components as a tool to discriminate between organic and conventional citrus fruits

The aim of this work was to develop a method for authenticity control of organically grown orange fruits. Due to the different kinds of nitrogen fertilization of the soil in organically and conventionally managed farms, the study tried to verify the possibility to differentiate Navelina and Tarocco orange fruits obtained by these production systems through the detection of markers linked to nitrogen metabolism. In addition to the classic quality parameters, total nitrogen (N) and synephrine contents in juice and (15)N/(14)N isotope ratio (expressed as delta(15)N per thousand) in proteins of pulp and amino acids of juice were determined. The results obtained indicated that total N and synephrine contents were significantly higher in conventional fruits, whereas the delta(15)N per thousand values were higher in the organic ones. The new markers identified in this research by linear discriminant analysis of the data may constitute a useful tool to differentiate organic citrus fruits or juices from conventional ones.     

A molecular basis for natural selection at the timeless locus in Drosophila melanogaster

Diapause is a protective response to unfavorable environments that results in a suspension of insect development and is most often associated with the onset of winter. The ls-tim mutation in the Drosophila melanogaster clock gene timeless has spread in Europe over the past 10,000 years, possibly because it enhances diapause. We show that the mutant allele attenuates the photosensitivity of the circadian clock and causes decreased dimerization of the mutant TIMELESS protein isoform to CRYPTOCHROME, the circadian photoreceptor. This interaction results in a more stable TIMELESS product. These findings reveal a molecular link between diapause and circadian photoreception.     

Midbody targeting of the ESCRT machinery by a noncanonical coiled coil in CEP55

The ESCRT (endosomal sorting complex required for transport) machinery is required for the scission of membrane necks in processes including the budding of HIV-1 and cytokinesis. An essential step in cytokinesis is recruitment of the ESCRT-I complex and the ESCRT-associated protein ALIX to the midbody (the structure that tethers two daughter cells) by the protein CEP55. Biochemical experiments show that peptides from ALIX and the ESCRT-I subunit TSG101 compete for binding to the ESCRT and ALIX-binding region (EABR) of CEP55. We solved the crystal structure of EABR bound to an ALIX peptide at a resolution of 2.0 angstroms. The structure shows that EABR forms an aberrant dimeric parallel coiled coil. Bulky and charged residues at the interface of the two central heptad repeats create asymmetry and a single binding site for an ALIX or TSG101 peptide. Both ALIX and ESCRT-I are required for cytokinesis, which suggests that multiple CEP55 dimers are required for function.