Biopterin Status in Dogs with Myxomatous Mitral Valve Disease is Associated with Disease Severity and Cardiovascular Risk Factors

Background

Endothelial dysfunction (ED) has been suggested to be associated with myxomatous mitral valve disease (MMVD) in dogs. Tetrahydrobiopterin (BH4) is an important cofactor for production of the endothelium‐derived vasodilator nitric oxide (NO). Under conditions of oxidative stress, BH4 is oxidized to the biologically inactive form dihydrobiopterin (BH2). Thus, plasma concentrations of BH2 and BH4 may reflect ED and oxidative stress.

Objective

To determine plasma concentrations of BH2 and BH4 in dogs with different degrees of MMVD.

Animals

Eighty‐four privately owned dogs grouped according to ACVIM guidelines (37 healthy control dogs including 13 Beagles and 24 Cavalier King Charles Spaniels [CKCSs], 33 CKCSs with MMVD of differing severity including 18 CKCSs [group B1] and 15 CKCSs [group B2], and 14 dogs of different breeds with clinical signs of congestive heart failure [CHF] because of MMVD [group C]).

Methods

Dogs underwent clinical examination including echocardiography. Plasma concentrations of BH2 and BH4 were measured using high‐performance liquid chromatography with fluorescence detection.

Results

Higher plasma BH4 and BH2 concentrations were found with dogs in CHF compared with all other groups (control, B1 and B2; P ≤ .001). Females had higher concentrations of BH4 and BH4/BH2 (P ≤ .0003). BH4/BH2 was found to decrease with age (P < .0001). Cardiovascular risk factors in humans such as passive smoking (P ≤ .01) and increased body weight (P ≤ .009) were associated with lower BH4 concentrations.

Conclusions and Clinical Importance

Age, sex, body weight, passive smoking, and cardiac status are associated with plasma biopterin concentration in dogs. Additional studies should clarify the clinical implications of the findings.     

Differentiation of important and closely related cereal plant species (Poaceae) in food by hybridization to an oligonucleotide array

We report the development of an oligonucleotide microarray for the simultaneous detection of six important cereal food plant species from the Poaceae based on the chloroplast trnL intron sequence. We used universal primers to amplify the trnL intron from wheat, rye, barley, oat, rice, and maize, followed by a cyclic labeling of oligonucleotides probes and subsequent hybridization to an oligonucleotide microarray. In single taxon analyses, positive signals were produced with a high signal-to-noise ratio. The assay also enabled the analysis of mixed samples. The results obtained for real food samples were in agreement with the ingredient labels, but positive results for grains not declared on the ingredients list were observed in three out of 10 samples, which indicates that the final products and/or the declared ingredients were probably botanically impure or contaminated. The combination of the sensitivity of a universal polymerase chain reaction with the specificity of the labeling reaction allows this protocol to be applied in routine analyses of food samples, as demonstrated by successful analysis of processed composite food products.     

Comparative genotoxicity testing of rhine river sediment extracts using the comet assay with permanent fish cell lines (rtg-2 and rtl-w1) and the ames test*

Goals, Scope and Background

Improved quality of surface waters and sediments requires advanced strategies for ecotoxicological assessment. Whilst at least in Germany assessment strategies on the basis of chemical analysis and acute toxicity data dominated the last decades, the development of more specific biological endpoints and biomarkers in ecotoxicology is required in order to arrive at a good ecological potential and good chemical status of surface waters in the European river basins until the year 2015, as required by the European Water Framework Directive. Since sediments have for long been known to function both as a sink and as a source of pollutants in aquatic systems, and since part of the particle-associated substances have frequently been demonstrated to cause mutagenic and carcinogenic effects in aquatic organisms, particularly in fish, there is, among other requirements, an urgent need to develop, standardize and implement integrated vertebrate-based test systems addressing genotoxicity into recent sediment investigation strategies. Thus, the present study was designed to compare the suitability of two commonly used test systems, the comet assay and the Ames test, for the evaluation of the ecotoxicological burden of surface and core sediment samples from the river Rhine.

Methods (or Main Features)

In order to determine the importance of inherent enzymatic activities, two permanent fish cell lines with different biotransformation capacities, RTL-W1 and RTG-2, were compared with respect to their capability of detecting genotoxic effects in 18 surface and core sediment samples from 9 locations along the River Rhine in the comet assay with and without exogenous bioactivation. For further comparison, as a prokaryotic mutagenicity assay, theSalmonella plate incorporation assay (Ames test) with the test strains TA98 and TA 100 with and without exogenous metabolic activation was used.

Results and Discussion

Whereas all sediment extracts induced genotoxic effects in the comet assay with RTL-W1 cells, only 12 out of 18 sediment extracts revealed significant genotoxicity in the tests with the less biotransformation-competent RTG-2 cells. Exogenous bioactivation by addition of ß-naphthoflavone /phenobarbital-induced S9 from rat liver resulted in both reduction or increase of genotoxicity in samples from different sites, however, without consistent reaction patterns. In general, the responses of RTL-W1 cells indicated higher biotransformation capacity than in RTG-2 cells without S9 complementation. In Ames tests using TA98 with S9, 16 out of 18 extracts induced significant mutagenicity with induction factors up to 4. Compared to TA98, the strain TA100 proved less sensitive, with maximum induction factors of 1.3, indicating the potential presence of substances inducing frarneshift mutations, which can only be detected in the strain TA98. Chemical analyses revealed particularly high levels of hexachlorbenzene (up to 860 µg/kg) and priority PAHs (up to 4.8 mg/kg); so far, however, no correlation could be found between compounds analyzed and the corresponding biotests.

Conclusions

Results document that both comet assay and Ames test are capable of detecting xenobiotic interaction with DNA in consequence of exposure to complex environmental samples. Whereas the alkaline version of the comet assay detects a broad range of interactions with the DNA, however without information about their eventual importance, the Ames test only reveals established mutations, but fails to detect transient (reparable) DNA alterations. However, even transient primary changes in the DNA structure might result in carcinogenic processes and, eventually, in implications at the population level. As a consequence, for hazard assessment purposes, a combination of both assays is required to avoid false negatives in genotoxicity evaluation. Poor correlation between data obtained by chemical analysis and results in bioassays is indicative of our limited understanding of the sources of genotoxicity. In fact, numerous studies combining chemical and biological approaches for hazard assessment of complex environmental mixtures indicate that priority pollutant concentrations are a poor indicator of toxicity.If compared to the cell line RTG-2, RTL-W1 proved more effective in detecting genotoxicity in surface sediment samples and, thus, indicated the importance of bioactivation of at least part of the compounds in superficial layers of sediments. Results further document that the common assumption may be wrong that, in comparison to deeper strata, surface layers carry a lower toxic burden in consequence of the current decrease in water pollution. This might at least in part be due to remobilization of more heavily polluted sediments from deeper layers during severe flood events followed by re-sedimentation in flood plains or upstream weirs, where they might cover less polluted younger sediment layers.

Recommendations and Perspectives

For a comprehensive assessment of genotoxicity in surface and core sediments, a combination of eukaryotic (comet assay) and prokaryotic assays (Ames test) with and without exogenous bioactivation is recommended. Since studies with organic sediments extracts simulate a worst-case scenario and fail to take into account bioavailability, there is broad consensus that whole-sediment exposure protocols represent the most realistic scenarios. Whereas more realistic solid phase exposure has frequently been applied in both microbial and invertebrate acute toxicity testing, there is an urgent need to develop corresponding whole sediment fish-based genotoxicity tests.

     

Immunohistochemical and Taqman real-time PCR detection of mycobacterial infections in fish

Real‐time PCR and immunohistochemistry (IHC) assays were developed to detect fish mycobacterial infections at the genus level, based on the RNA polymerase β subunit (rpoB) gene and polyclonal anti‐Mycobacterium rabbit serum, respectively. The PCR assay positively identified a number of pathogenic mycobacteria including Mycobacterium abscessus, M. avium ssp. avium, M. bohemicum, M. chelonae ssp. chelonae, M. farcinogenes, M. flavescens, M. fortuitum ssp. fortuitum, M. gastri, M. gordonae, M. immunogenicum, M. malmoense, M. marinum, M. montefiorense, M. phlei, M. phocaicum, M. pseudoshottsii, M. salmoniphilum, M. senegalense, M. shottsii, M. smegmatis, M. szulgi and M. wolinskyi. A detection limit equivalent to 10² cfu g^?1 was registered for M. salmoniphilum‐infected fish tissue. The IHC precisely localized both free and intracellular mycobacteria in tissues and detected mycobacterial infections down to 10² cfu g^?1 tissue. Both assays were found to be more sensitive than Ziehl–Neelsen (ZN) staining, where the detection limit was below 8 × 10³ cfu g^?1 tissue. Although specificity testing of the real‐time PCR against a panel of non‐Mycobacterium spp. revealed a degree of cross‐reaction against pure DNA extracted from Nocardia seriolae and Rhodococcus erythropolis, no cross‐reactions were identified (by either real‐time PCR or IHC) on testing of formalin‐fixed paraffin‐embedded (FFPE) tissues confirmed to be infected with these bacteria. The broad applicability of both assays was confirmed by analysis of FFPE tissues from a range of fish species infected with diverse Mycobacterium spp. The results indicate that both assays, alone or in combination, constitute sensitive tools for initial, rapid diagnosis of mycobacteriosis in fish. This should in turn allow rapid application of more specific studies, i.e. culture based, to identify the specific Mycobacterium sp. involved.     

Tagging of juvenile lobsters (Homarus gammarus (L.)) with visible implant fluorescent elastomer tags

Visible implant fluorescent elastomer (VIFE) tags were implanted beneath the epidermal layer within the abdomen of 25 juvenile lobsters. After three molts the tag retention was 100% and the total survival 92%. The results suggest that VIFE tags could be an effective tool when assessing the feasibility of enhancing natural lobster stocks.     

Persistent effects of fire severity on early successional forests in interior Alaska

There has been a recent increase in the frequency and extent of wildfires in interior Alaska, and this trend is predicted to continue under a warming climate. Although less well documented, corresponding increases in fire severity are expected. Previous research from boreal forests in Alaska and western Canada indicate that severe fire promotes the recruitment of deciduous tree species and decreases the relative abundance of black spruce (Picea mariana) immediately after fire. Here we extend these observations by (1) examining changes in patterns of aspen and spruce density and biomass that occurred during the first two decades of post-fire succession, and (2) comparing patterns of tree composition in relation to variations in post-fire organic layer depth in four burned black spruce forests in interior Alaska after 10-20 years of succession. We found that initial effects of fire severity on recruitment and establishment of aspen and black spruce were maintained by subsequent effects of organic layer depth and initial plant biomass on plant growth during post-fire succession. The proportional contribution of aspen (Populus tremuloides) to total stand biomass remained above 90% during the first and second decades of succession in severely burned sites, while in lightly burned sites the proportional contribution of aspen was reduced due to a 40-fold increase in spruce biomass in these sites. Relationships between organic layer depth and stem density and biomass were consistently negative for aspen, and positive or neutral for black spruce in all four burns. Our results suggest that initial effects of post-fire organic layer depths on deciduous recruitment are likely to translate into a prolonged phase of deciduous dominance during post-fire succession in severely burned stands. This shift in vegetation distribution has important implications for climate-albedo feedbacks, future fire regime, wildlife habitat quality and natural resources for indigenous subsistence activities in interior Alaska.     

Comparison of a DNA probe, complement-fixation and indirect immunofluorescence tests for diagnosing Anaplasma marginale in suspected carrier cattle

Most estimates of the prevalence of anaplasmosis have been based on serologic data using the complement-fixation (CF) and/or card agglutination tests. Since these tests are considered to be only about 50 percent reliable for detecting carrier cattle in enzootically stable herds, the need for more sensitive diagnostic tests is widely recognized. The objective in the present study was to compare the sensitivity of the CF test with that of the indirect immunofluorescence (IIF) test and a recently developed DNA probe in determining the prevalence of Anaplasma marginale infection in cattle from an enzootic area. The study herd consisted of 52 8-month-old steers and 13 3-year-old cows of mixed beef breed. All cattle were initially tested for this comparative purpose. All but one animal (one that was a positive reactor as assessed by all three tests, and served as a positive control), were treated with long-acting oxytetracycline in an attempt to clear any carrier infections. Each animal was then retested at 1 month and 2 months post-treatment (PT), in an effort to determine if the DNA probe could be used to evaluate the effectiveness of the drug. Six of the 65 (9.2%) initial serum samples were CF positive. In contrast, 60 (92.3%) and 64 (98.5%) of the samples were positive as assessed with the IIF test and the DNA probe, respectively. The DNA hybridization reactions varied in intensity within the sample population indicating different individual levels of infection. The DNA probe hybridized with two samples taken at 1 month PT, and with two different samples taken at 2 months PT. The mean IIF titers were reduced at both the 1 month and 2 month sampling times. These results suggest that the drug did not eliminate infections in all cattle. Some may have been cleared, but, in any event, the drug did reduce the level of infections below the sensitivity of the DNA probe and interrupted continuity of stimulation of antibody. Therefore, the DNA probe and the IIF test appear to be considerably more sensitive in detecting carrier infections than the CF test, and should be considered in future epidemiologic studies.