Involvement of cytolytic and non-cytolytic T cells in the control of feline immunodeficiency virus infection

The appearance of non-cytolytic T cells that suppressed feline immunodeficiency virus (FIV) replication in vitro, and FIV-specific cytotoxic T cell (CTL) responses was compared in a group of seven, specific pathogen free (SPF) domestic cats following primary infection with the Glasgow(8) isolate of FIV (FIV(GL-8)). FIV proviral burdens were quantified in the blood and lymphoid tissues by real-time PCR. Non-cytolytic T cell suppression of FIV replication was measured by co-cultivating lymphoblasts prepared from the cats at different time-points during infection with FIV-infected MYA-1 cells in vitro. Non-cytolytic suppressor activity was detected as early as 1 week after infection, and was evident in all the lymphoid tissues examined. Further, this activity was present in subpopulations of T cells in the blood with normal (CD8(hi)) or reduced (CD8(lo)) expression of the CD8 molecule, and temporal modulations in non-cytolytic suppressor activity were unrelated to the circulating CD8(+) T cell numbers. Virus-specific CTL responses, measured by (51)Cr release assays, were not detected until 4 weeks after infection, with the emergence of FIV-specific effector CTLs in the blood. Throughout infection the response was predominantly directed towards FIV Gag-expressing target cells, and by 47 weeks after infection CTL responses had become localised in the lymph nodes and spleen. The results suggest that both non-cytolytic T cell suppression of FIV replication and FIV-specific CTL responses are important cellular immune mechanisms in the control of FIV replication in infected asymptomatic cats.     

An Echocardiographic Study of Atrial Fibrillation in Horses: Before and After Conversion to Sinus Rhythm

Two-dimensional and M-mode echocardiograms were recorded from 41 horses before they were successfully treated for atrial fibrillation. In addition, these examinations were performed in a subgroup of 20 horses after treatment, and the results were compared with pretreatment values. Atrial fibrillation in this group of horses was associated with a reduction of mean left ventricular fractional shortening (mean 31 %± 5.24%), and 22 of the 41 horses were below the reference range. The remaining mean M-mode variables were within the normal reference range, although 12 horses had increased left ventricular lumen dimensions in systole, and 8 horses had decreased left ventricular ejection times. Abnormal motion of the mitral valve was present in all horses and was characterized by the absence of A peaks, which were replaced by small diastolic undulations in 55% of the horses. In horses 1 to 20, after conversion to sinus rhythm, the mean fractional shortening increased (35.34%± 5.4%, P = .004), but there were no significant differences in heart rate or left ventricular lumen diameters in systole or diastole. These results suggest that ventricular function may be compromised by the presence of atrial fibrillation. However, this improved after correction of the arrhythmia.     

Characterization of a bacteriophage lysin (Ply700) from Streptococcus uberis

The antibacterial properties of bacteriophage lytic enzymes may be of importance in future mastitis control programs. A prophage was isolated from a strain of Streptococcus uberis (ATCC 700407) following exposure to mitomycin C. Partial sequencing of the phage DNA revealed a putative lysin based on sequence similarity to other streptococcal phage lysins. The putative lysin (Ply700) was recombinantly expressed in Escherichia coli, and chromatographically purified. Addition of the purified Ply700 to bacterial suspensions of S. uberis, Streptococcus pyogenes, and Streptococcus dysgalactiae caused a rapid, calcium-dependent lysis while there was little activity against Streptococcus agalactiae, Staphylococcus aureus, or E. coli. Killing of S. uberis in milk by Ply700 (50 microg/ml) was confirmed by plate count assay. Activity was related to the initial concentration of bacteria in that 31% killing (P<0.05) was observed with an inoculating dose of approximately 4500 cfu/ml, while 81% killing (P<0.01) was observed when the inoculum was reduced to approximately 600 cfu/ml. In contrast, complete sterilization was observed in parallel cultures suspended in assay buffer indicating that factors in milk are able to neutralize the lysin. Functional characterization of the C-terminal domain, as a component of a GFP fusion protein, revealed its calcium-dependent ability to bind to S. uberis. The C-terminal domain may have utility in targeting S. uberis while it remains to be determined if the lysin by itself has sufficient potency in milk for effective use in the control of S. uberis mastitis.     

Repair of oronasal fistulae using auricular cartilage grafts in five cats

OBJECTIVE: To describe repair of chronic palatine defects in cats, with free cartilage graft harvested from either the pinna (scapha) or vertical ear canal (annular cartilage). STUDY DESIGN: Retrospective study. ANIMALS: Cats (n=5) with chronic oronasal fistula. METHODS: Cartilage was harvested from either the scapha or annular cartilage and epithelium removed. After preparation of the edges of the palatal defect by separation of the oral and palatal mucosa circumferentially, the graft was inserted between the epithelial layers and secured without tension. The graft acted as a scaffold for migration of granulation tissue and epithelialization. RESULTS: Oronasal fistulae were successfully repaired in 5 cats (3 pinna grafts; 2 annular cartilage grafts); 1 cat required a second graft after the first graft was dislodged. CONCLUSIONS: Auricular cartilage provides a reliable framework for repair of oronasal fistulae in cats. CLINICAL RELEVANCE: Chronic oronasal fistulae refractory to conventional repair can be treated by use of auricular free graft (pinna or annular cartilage) without disfigurement. Use of annular cartilage to support palatal repairs offers surgeons an additional option when other methods have failed. We recommend using conchal cartilage as the initial treatment approach for repair of small oronasal fistulae in cats.     

Roads as sources of heavy metals in urban areas. The Covões catchment experiment,Coimbra, Portugal

Purpose

This work studies the implications of different traffic patterns for heavy metal and solid pollution generation processes following rainfall events with contrasting antecedent meteorological conditions, at a periurban catchment. The aim is to provide information on the pollution processes and their potential environmental impacts for urban areas.

Materials and methods

Seven campaigns were performed covering winter, spring, and summer conditions, for rainfall events with different antecedent conditions. Four types of roads were monitored: low traffic, average traffic, heavy traffic with demanding driving situations (break and turning), and heavy traffic with high vehicle speed (motorway profile). Samples were taken at the beginning, middle and end of the events to measure within event variation in concentration. Analytical standard procedures were used to quantify pH, conductivity, turbidity, total solids, volatile solids, suspended solids, volatile suspended solids and heavy metals (Cd, Cu, Pb and Zn) in the total and dissolved forms (as to infer the particulate fraction), namely copper, zinc, cadmium and lead.

Results and discussion

The collected data show a direct relation among the number of vehicles and/or the driving manoeuvres performed by them and the amount of solids and heavy metals present in the wash out overland flow collected. An important fraction of the heavy metals is washed off in the particulate form, which represents an increased problem since the road overland flow is directed to green/brown areas and for the local aquatic ecosystems. Maximum copper values recorded exceed 0.6 mh L^?1, zinc exceeds 5 mg L^?1, lead 0.1 mg L^?1 and cadmium 0.01 mg L^?1. Values are higher after long dry spells and reduce concentration throughout the rainfall events.

Conclusions

An important part of the heavy metals (with relevance for zinc and lead) are washed off in the particulate form, pollutants are typically related to the amount of traffic, and especially to the existence of driving manoeuvres. The summer events show the highest values, due to the accumulation of pollutants during the long dry spells.

     

Mini-cuttings: an effective technique for the propagation of <Emphasis Type="Italic">Pinus pinaster</Emphasis> Ait.

Pinus pinaster Ait. is one of the main forest tree species planted in Spain, Portugal and France. Due to its high economic relevance, there is considerable interest in developing techniques for vegetative breeding aimed at mass propagation. In this study we present a mini-propagation protocol in order to define an efficient method to propagate families or clones of P. pinaster. We carried out three experiments using mini-cuttings of 3–5 cm in length with the aim of evaluating the effects of temperature (4°C vs. 25°C), plant growth regulator (IBA) and shoot age on rooting ability. Percentage of rooted cuttings and morphological root variables were recorded. The percentage of rooted cuttings per treatment ranged from 68 to 97%. Treatment with IBA significantly influenced the rooting process at 25°C but not at 4°C. The number of apexes, length, area and volume of roots were all positively affected by temperature treatment. Shoot age also had a positive effect on rooting capacity of cuttings, with the cuttings from the youngest shoots (70 days after pruning) having higher rooting percentages, ranging from 84.7 to 98.3%. The use of juvenile material, good environmental conditions and IBA all benefited the rooting of clonal material, resulting in high rooting capacity. This study presents an innovative propagation protocol for P. pinaster that can be used as a tool in breeding programs.     

Hapten synthesis and monoclonal antibody-based immunoassay development for the detection of the fungicide kresoxim-methyl

Strobilurin fungicides have been increasingly used for fungus pest control since they were introduced in 1996. For pesticide residue detection, immunoassays constitute nowadays a valuable approach. This paper describes the synthesis of functionalized haptens of kresoxim-methyl, the production of monoclonal antibodies, and the development of enzyme-linked immunosorbent assays. On the one hand, a two-step conjugate-coated immunoassay was optimized using extended or short incubation times, with limits of detection of 0.4 ng/mL for the extended assay and 0.3 ng/mL for the rapid assay. On the other hand, an immunoassay was optimized following a procedure consisting of just one incubation step. This one-step assay had a limit of detection of 0.4 ng/mL. All of these assays showed a similar performance, with sensitivities well below common maximum residue limits for this pesticide (50 microg/kg) and lower than the detection limits of the usual chromatographic detection methods.     

Production and characterization of monoclonal antibodies specific to the strobilurin pesticide pyraclostrobin

Strobilurin fungicides are nowadays among the most important fungicides in the market of active agrochemicals. Pyraclostrobin, which belongs to the last generation of this family of molecules, shows a broader antifungal activity spectrum and higher efficiency and security profiles than previous fungicides. This paper describes the synthesis of functionalized haptens, the production of monoclonal antibodies, and the development of enzyme-linked immunosorbent assays (ELISA) for the detection of pyraclostrobin. A conformational analysis of hapten structure was performed, which provided relevant data concerning the length of the spacer arm. A very useful strategy has been followed for the screening of hybridomas, leading to the selection of a panel of high-affinity monoclonal antibodies to pyraclostrobin. Moreover, different immunoassays have been characterized using the conjugate-coated indirect ELISA format, and limits of detection below 0.1 microg/L have been obtained. Also, a simplified one-step procedure has been carried out with two indirect assays. Finally, these results have been compared with the performance of the same antibodies in the antibody-coated direct ELISA format.     

Effect of the simultaneous addition of beta-cyclodextrin and the herbicide norflurazon on its adsorption and movement in soils

The effects of beta-cyclodextrin (BCD) on the sorption-desorption and transport processes of the herbicide norflurazon (NFL) in soils of different characteristics when both are applied simultaneously have been investigated. Adsorption-desorption studies of NFL on six soils of very different characteristics in the presence of BCD have been performed using a batch equilibration method and correlated to its mobility in homogeneous hand-packed soil columns. NFL determinations were undertaken by HPLC equipped with a diode array detector at a wavelength of 220 nm. BCD was also analyzed by HPLC with fluorimetric detection using a postcolumn reaction. The interaction of NFL with BCD yielded the formation of an inclusion complex in solution. When this complex is applied to soils, a large decrease in NFL adsorption capacity and an increase in its desorption were observed, due to the higher tendency of NFL-BCD complexes to remain in solution. The results obtained in adsorption and soil column experiments indicated that the influence of BCD on NFL mobility and availability depends on the different affinities of BCD to be sorbed on soils of different characteristics and on the concentration of BCD used. The lower the concentration of BCD added, the more tenaciously it adheres to the soil, and most of the BCD molecules would be adsorbed, providing a coating to soil particles that acts as a bridge between NFL and the soil surface, acting as an adsorbent and retarding the mobility of the herbicide. At higher concentrations of BCD, or in soils where its adsorption is very low, most of the BCD molecules are in the aqueous phase and NFL molecules tend to be complexed with BCD in solution, acting then as a solubilizing agent.