Expression of inducible nitric oxide synthase in macrophages inversely correlates with parasitism of lymphoid tissues in dogs with visceral leishmaniasis

Background

There are only a few studies reporting the role of nitric oxide metabolites for controlling macrophage intracellular parasitism, and these are controversial. Therefore, the present study aimed to evaluate the expression of inducible nitric oxide synthase (iNOS) in the lymph nodes and spleen of dogs affected by visceral leishmaniasis through immunohistochemistry and to determine its correlation with tissue parasite burden and serum interferon (IFN)-γ levels. Twenty-eight dogs were selected and assigned to one of two groups, symptomatic (n = 18) and asymptomatic (n = 10), according to clinical status and laboratory evaluation. A negative control group (n = 6) from a non-endemic region for visceral leishmaniasis was included as well.

Results

Parasite density (amastigotes/mm²) was similar between clinical groups in the lymph nodes (P = 0.2401) and spleen (P = 0.8869). The density of iNOS⁺ cells was higher in infected dogs compared to controls (P < 0.05), without a significant difference in lymph node (P = 0.3257) and spleen (P = 0.5940) densities between symptomatic and asymptomatic dogs. A positive correlation was found between the number of iNOS⁺ cells in lymph nodes and interferon-γ levels (r = 0.3776; P = 0.0303), and there was a negative correlation between parasites and iNOS⁺ cell densities both in lymph nodes (r = −0.5341; P = 0.0034) and spleen (r = −0.4669; P = 0.0329).

Conclusion

The negative correlation observed between tissue parasitism and the expression of iNOS may be a reflection of NO acting on the control of parasites.     

Saw Palmetto (Serenoa repens) Flowering and Fruiting Response to Time Since Fire

Saw palmetto (Serenoa repens [Bartr.] Small) is a shrubby palm common in southeastern US pine flatwoods ecosystems. Demand recently has increased for fruits for the herbal remedies market. Because only wild saw palmettos are harvested, management strategies are needed to promote flowering and fruiting. This study investigated effects of time since growing season (April–July) fires on flowering and fruiting of saw palmetto ramets ≥ 54 cm in height, in 18 pine flatwoods or dry prairie sites (six sites in three locations, burned in 1996, 1995, 1994, 1993, 1992, or before 1991) in central and southwest Florida from 1996 to 1999. We used repeated measures, linear mixed models to test for time since fire effects on proportion of ramets flowering, proportion of ramets fruiting, and fruit yield. Ranges of means among sites over all years of the study for proportion of ramets flowering, proportion of ramets fruiting, and fruit yield were 0 to 0.78, 0 to 0.72, and 0 kg · ha^?1 to 2 869 kg · ha^?1, respectively. Time since fire strongly influenced flowering; highest probability of flowering occurred 1 yr after burning, followed by an abrupt decrease 2 yr after burning, then a gradual increase from 3 to 5 yr after fires (polynomial regression, P < 0.0001 for fixed effects). Probability of fruiting increased with increasing time since fire (quadratic regression, P < 0.001 for fixed effects), but fruit yields showed no pattern in response to time since fire (P=0.916). The decrease in influence of fire from flowering through fruit maturity presumably was caused by mortality from factors such as caterpillar predation and fungal infection. To promote increased flowering and fruit yields, we recommend that growing season burns be conducted approximately every 5 yr. We suggest, however, that management strategy be modified as necessary to maintain ecosystem diversity and function.     

Investigations on the efficacy of routinely used phenotypic methods compared to genotypic approaches for the identification of staphylococcal species isolated from companion animals in Irish veterinary hospitals

Background

Identification of Staphylococci to species level in veterinary microbiology is important to inform therapeutic intervention and management. We report on the efficacy of three routinely used commercial phenotypic methods for staphylococcal species identification, namely API Staph 32 (bioMérieux), RapID (Remel) and Staph-Zym (Rosco Diagnostica) compared to genotyping as a reference method to identify 52 staphylococcal clinical isolates (23 coagulase positive; 29 coagulase negative) from companion animals in Irish veterinary hospitals.

Results

Genotyping of a 412 bp fragment of the staphylococcal tuf gene and coagulase testing were carried out on all 52 veterinary samples along with 7 reference strains. In addition, genotyping of the staphylococcal rpoB gene, as well as PCR-RFLP of the pta gene, were performed to definitively identify members of the Staphylococcus intermedius group (SIG). The API Staph 32 correctly identified all S. aureus isolates (11/11), 83% (10/12) of the SIG species, and 66% (19/29) of the coagulase negative species. RapID and Staph-Zym correctly identified 61% (14/23) and 0% (0/23) respectively of the coagulase-positives, and 10% (3/29) and 3% (1/29) respectively of the coagulase-negative species.

Conclusions

Commercially available phenotypic species identification tests are inadequate for the correct identification of both coagulase negative and coagulase positive staphylococcal species from companion animals. Genotyping using the tuf gene sequence is superior to phenotyping for identification of staphylococcal species of animal origin. However, use of PCR-RFLP of pta gene or rpoB sequencing is recommended as a confirmatory method for discriminating between SIG isolates.     

Effects of Ω-3 (n-3) Fatty Acid Supplementation on Insulin Sensitivity in Horses

The objective of this study was to examine the effects of dietary ω-3 fatty acid supplementation on insulin sensitivity (SI) in horses. Twenty-one mares were blocked by age, body weight (BW), and body condition score (BCS) and randomly assigned to one of three dietary treatments. Treatments consisted of (1) 38 g of n-3 fatty acids via fish and algae supplement and diet (MARINE), (2) 38 g of n-3 fatty acids via a flaxseed meal from the supplement and diet (FLAX), and (3) control (CON) no supplemental fatty acid. Treatments were supplemented for 90 days. Frequent sampling intravenous glucose tolerance tests were performed on days 0, 30, 60, and 90. Blood samples were analyzed for glucose and insulin. The minimal model was applied for the glucose and insulin curves using MinMod Millennium. SI increased 39% (P < .007) across all treatment groups. Acute insulin response to glucose decreased 22% (P < .006) between days 30 and 60 and increased (P = .040) again at day 90. Disposition index (combined SI and β pancreatic response) increased (P = .03) by 53% in the MARINE- and 48% in the FLAX-supplemented horses and did not change with time in the CON group. In insulin-resistant mares, MARINE- and FLAX-treated horses had an increase in SI (P = .09). It would be interesting to test this supplement in a larger group of insulin-resistant horses. If proven effective, supplementation with ω-3 fatty acids would help to reduce problems associated with insulin resistance in horses.     

Phytochemical composition and metabolic performance-enhancing activity of dietary berries traditionally used by Native North Americans

Four wild berry species, Amelanchier alnifolia, Viburnum trilobum, Prunus virginiana, and Shepherdia argentea, all integral to the traditional subsistence diet of Native American tribal communities, were evaluated to elucidate phytochemical composition and bioactive properties related to performance and human health. Biological activity was screened using a range of bioassays that assessed the potential for these little-known dietary berries to affect diabetic microvascular complications, hyperglycemia, pro-inflammatory gene expression, and metabolic syndrome symptoms. Nonpolar constituents from berries, including carotenoids, were potent inhibitors of aldose reductase (an enzyme involved in the etiology of diabetic microvascular complications), whereas the polar constituents, mainly phenolic acids, anthocyanins, and proanthocyanidins, were hypoglycemic agents and strong inhibitors of IL-1beta and COX-2 gene expression. Berry samples also showed the ability to modulate lipid metabolism and energy expenditure in a manner consistent with improving metabolic syndrome. The results demonstrate that these berries traditionally consumed by tribal cultures contain a rich array of phytochemicals that have the capacity to promote health and protect against chronic diseases, such as diabetes.     

Optimization of lycopene extraction from tomato cell suspension culture by response surface methodology

Radioisotope-labeled lycopene is an important tool for biomedical research but currently is not commercially available. A tomato cell suspension culture system for the production of radioisotope-labeled lycopene was previously developed in our laboratory. In the current study, the goal was to optimize the lycopene extraction efficiency from tomato cell cultures for preparatory high-performance liquid chromatography (HPLC) separation. We employed response surface methodology (RSM), which combines fractional factorial design and a second-degree polynomial model. Tomato cells were homogenized with ethanol, saponified by KOH, and extracted with hexane, and the lycopene content was analyzed by HPLC-PDA. We varied five factors at five levels: ethanol volume (1.33-4 mL/g); homogenization period (0-40 s/g); saturated KOH solution volume (0-0.67 mL/g); hexane volume (1.67-3 mL/g); and vortex period (5-25 s/g). Ridge analysis by SAS suggested that the optimal extraction procedure to extract 1 g of tomato cells was at 1.56 mL of ethanol, 28 s homogenization, 0.29 mL of KOH, 2.49 mL of hexane, and 17.5 s vortex. These optimal conditions predicted by RSM were confirmed to enhance lycopene yield from standardized tomato cell cultures by more than 3-fold.     

Earthworm populations in septic system filter fields and potential effects on wastewater renovation

Wastewater renovation in septic-system filter fields can be affected by preferential flow through soil macropores. Anecic earthworm species make deep vertical burrows that may reduce renovation by acting as preferential flow paths that decrease effluent contact with the soil matrix. On the other hand, endogeic earthworms make largely horizontal burrows that may enhance wastewater renovation by distributing the effluent over a larger area. Additionally, the moist, nutrient-rich environment in filter fields may increase earthworm populations by enhancing their survival. Therefore, our objectives were to determine earthworm numbers and biomass with distance from soil treatment trenches, and identify species present to estimate potential effects on wastewater renovation. Five septic systems were investigated. At each site, earthworm populations were measured using formalin extraction at 10 locations along each of three 7-m long transects perpendicular to the trenches. There were an average of 6.4 times more earthworms and 5.4 times more earthworm biomass within 1 m of the trench than in the background (3.5–7.0 m from the trenches) in 13 of the 15 transects. This suggests that earthworms may have a significant effect on the movement of effluent. Because only epigeic and endogeic species were observed, the potential for reduced renovation and groundwater contamination at these sites is likely low. This may not be the case in areas with large numbers of anecic earthworms.     

The Effects of Feed Form on Consumption Time and Glucose and Insulin Response to a Concentrate Meal in Equine

This study tested the hypothesis that feeding an identically formulated, low sugar and starch concentrate in three forms (5-mm extruded [E], 4-mm pellet [P], and 19-mm oval [O]) would affect consumption rate and glucose or insulin responses, or both. Horses received 1.8 kg treatment feed in a randomized, crossover design, with samples taken every 30 minutes for 6 hours for blood glucose and insulin response. Pearson's correlation compared consumption time, insulin and glucose peak, and time to peak insulin and glucose. The pellet (P) elicited a lower (P = .01) glucose concentration at 2.5 hours than O. The pellet also elicited a lower (P = .03) insulin concentration at 5.5 hours than E and O. There were no differences (P > .05) in area under the curve (AUC) insulin, peak insulin, and time to peak insulin for the three treatments. Average insulin concentration was lower (P = .01) for P versus O. There were no differences (P > .05) in average insulin between P and E, nor between O and E. There were no differences (P > .05) in AUC and peak glucose concentration. Time to peak glucose was longer (P = .04) for P versus E. Average glucose concentration was lower (P = .02) for P versus O. Consumption time was longer (P = .03) for O versus P. There was a positive correlation between consumption time and time to peak insulin (r = 0.46, P = .029). Further research on feeding practices, feed forms, and consumption times that affect glycemic response is necessary.