Tissue basis for the use of enzymes in toxicology

The use of enzymes to monitor toxicological cell damage requires knowledge of: (1) the distribution of enzymes in the organs of the various animal species, (2) the variations of this distribution among cell types within a given organ, (3) the intracellular compartmentation of these enzymes. Moreover, kinetic parameters can also differ from one species to another.     

Gamma Glutamyl Transferase in domestic animals

In domestic animals, Gamma Glutamyl Transferase is mainly in the kidneys, the pancreas and the intestine; its liver activity is relatively high in cows, horses, sheep and goats and very low in dogs, cats and birds. The use of plasma reference values can help to interpret the variations of serum GGT mainly in hepatobiliary diseases of cattle, sheep, goats and cholestatic disorders of dogs. Urinary GGT is a good test of kidney toxic damage.     

Phytochemical and antioxidant analysis of eight Hypericum taxa from Central Italy

Eight taxa of the Hypericum spp. growing in Central Italy (Appennino Umbro-Marchigiano) were analyzed by HPLC-DAD for constituents quantitation, for antioxidant and free radical scavenging activities. H. perforatum subsp. veronense was the richest in phenolic compounds and hyperforin was detected for the first time in H. hircinum subsp. majus. Significant values of antioxidant activity were found in the investigated Hypericum taxa.     

Bartonella spp. DNA in cardiac tissues from dogs in Colorado and Wyoming

Background: Several Bartonella species (spp.) have been identified in dogs diagnosed with infectious endocarditis (IE) or myocarditis. Objective: To interrogate cardiac tissues of dogs with suspected IE for the presence of Bartonella spp. DNA of dogs in the Rocky Mountain states. Animals: Nine dogs with a clinical diagnosis of endocarditis from January 1990 to June 2008 were included. Methods: In this retrospective study, medical records at the Veterinary Teaching Hospital were searched. Animals were excluded if there was no diagnosis of IE in the original necropsy report. Paraffin embedded tissue blocks and medical records were available from 9 dogs. Total DNA was extracted from the cardiac tissues and assessed for Bartonella spp. DNA by 3 polymerase chain reaction (PCR) methods. For positive samples, the Bartonella spp. were determined by genetic sequencing or fluorogenic real‐time PCR. Results: Bartonella henselae DNA was amplified from the tissues of 7 dogs; Bartonella vinsonii subsp berkhoffii DNA was amplified concurrently from 3 dogs. Six dogs were from Colorado and 1 was from Wyoming. Flea or tick infestations were reported in 2 dogs. Conclusions and Clinical Importance: Bartonella spp. should be on the differential list for dogs in the Rocky Mountain states. The results emphasize the need for routine use of external parasite control products even in regions perceived to have low risk for flea and tick infestations.     

An evaluation of fish health‐management practices and occupational health hazards associated with Pangasius catfish (Pangasianodon hypophthalmus) aquaculture in the Mekong Delta,Vietnam

This study aimed to evaluate the current status on the use of probiotics, disinfectants and antimicrobials in hatcheries, nurseries and grow‐out farms producing Pangasius catfish (Pangasianodon hypophthalmus) in the Mekong Delta, Vietnam. Semi‐structured interviews were conducted with 83 aquaculture enterprises (15 hatcheries, 32 nurseries and 36 grow‐out farms). Farmers reported use of a total of 24 different antimicrobials, e.g. for treatment of bacillary necrosis and motile aeromonad septicaemia, and a variety of disinfectants, probiotics and nutritional supplements. In contrast to small‐scale farmers, all large‐scale grow‐out farmers studied were certified and therefore had higher levels of formal education and specialized aquaculture training to diagnose and treat diseases. All farmers prepared their own medicated feed with a high risk of treatment failure, negative environmental impact from released antimicrobials and resistance development. Small‐scale farmers were at particular occupational health risks when handling antimicrobials and other chemicals, e.g. mixing medicated feed with bare hands. There is an urgent need to improve knowledge and use innovative approaches, e.g. private‐public partnerships, to assure a prudent use of chemicals, to improve capacity and access to disease diagnosis, particularly for small‐scale grow‐out farmers and nurseries. Efforts to control use of antimicrobials in aquaculture should be coordinated with the livestock and human health sectors taking an One‐Health approach.     

Influence of the dietary inclusion of <Emphasis Type="Italic">Gracilaria cornea</Emphasis> and <Emphasis Type="Italic">Ulva rigida</Emphasis> on the biodiversity of the intestinal microbiota of <Emphasis Type="Italic">Sparus aurata</Emphasis> juveniles

Partial substitutions of fish meal by 5, 15, or 25 % of Gracilaria cornea or Ulva rigida in experimental diets were evaluated to study their effects on biodiversity of intestinal microbiota composition in gilthead seabream (Sparus aurata) juveniles. The diets were offered to duplicate groups of 15 juvenile fish (14.0 ± 0.5 g) for 70 days, and at the end of the experiment the intestinal microbiota from four specimens of each treatment was analysed by denaturing gel gradient electrophoresis. Results showed that the substitution of fish meal by algae meal induced important modifications in the intestinal microbiota community, as a big reduction of the biodiversity when the highest percentage (25 %) of U. rigida was included. On the contrary, an increase on the number of species was detected when a 15 % of algae was included. Various Lactobacillus delbrueckii subspecies were selectively stimulated when G. cornea was included in the feed, and other bacterial species, such as those included in the Vibrio genus, were reduced.     

Clonal populations of Leptosphaeria maculans contaminating cabbage in Mexico

The race structure and genotypic diversity of four Leptosphaeria maculans populations isolated from Brassica oleracea (broccoli, cauliflower, cabbage, etc.) in central Mexico (Aguascalientes, Guanajuato and Zacatecas states) were analysed. Race structure was characterized by an unusually low diversity at three locations out of four. Fourteen minisatellite markers revealed a high proportion of repeated multilocus genotypes in these populations, combined with a significant linkage disequilibrium and strong clonal fraction (65–87%). The occurrence of the mating‐type idiomorphs always significantly departed from the 1:1 proportion expected in the case of random mating. Each population thus consists of a few (four to nine) multilocus genotypes which are specific to each location. These data strongly support the hypothesis of exclusive, or a high rate of, clonal multiplication. Comparison of cropping practices between B. oleracea and B. napus indicate that the shift in reproductive behaviour of the fungus is chiefly man‐mediated.     

Polymerase chain reaction fingerprinting of Erwinia amylovora has a limited phylogenetic value but allows the design of highly specific molecular markers

Erwinia amylovora, the causal agent of fire blight, is genetically very homogeneous, and current methodologies provide insufficient or contradictory information about the probable dispersal routes of the pathogen. With the final aim to obtain specific and reliable molecular markers for different lineages of the pathogen, we studied the molecular basis of rep-polymerase chain reaction (PCR) polymorphism using seven different arbitrary primers to fingerprint 93 E. amylovora strains from different countries, including Spain. Polymorphism was very low, and was displayed by only 11 E. amylovora strains, which produced 22 polymorphic bands. Five of 11 polymorphic bands cloned contained DNA that was present in more than 85% of the strains, whereas six bands were due to DNA present exclusively in the strains producing the rep-PCR polymorphism. Also, five of the polymorphic bands were due to the possession of either the ubiquitous plasmid pEA29, of plasmid pEU30, which was exclusively found in strains from North America, or of a 35-kb cryptic plasmid, present only in 28 strains from Northern Spain. We designed primer pairs from several cloned polymorphic bands that allowed the specific identification of the strains producing the polymorphism. Our results indicate that rep-PCR is not adequate for constructing genealogies of E. amylovora, although the strategy illustrated here, as well as the designed primers, can be used effectively in epidemiological studies with this pathogen.     

Iron exposure modifies acetylcholinesterase activity in zebrafish (Danio rerio) tissues: distinct susceptibility of tissues to iron overload

Iron is one the most abundant metals on the earth being essential for living organisms even though its free form can be toxic. The overload of this metal may be related with some disorders, like Alzheimer and Parkinson diseases, and hemochromatosis in the liver. The aim of the present study was to evaluate the effects of iron on acetylcholinesterase (AChE) activity in brain and liver of zebrafish and to investigate the possible correlation with the iron content in these tissues. Different corresponding concentrations of iron were tested using in vitro (0.018, 0.268, and 2.6 mM) and in vivo (1, 15, and 150 mg/l) assays. The in vitro studies showed that iron promoted a significant increase in AChE activity in brain (52%) and liver (53%) at the higher concentration (2.6 mM). In the in vivo assays, a significant increase in this enzyme activity was observed in the presence of 15 mg/l in both, brain (62%) and liver tissue (70%). Semiquantitative RT-PCR did not reveal significant changes in acetylthiocholinesterase mRNA levels. Moreover, we observed that iron content was significantly increased in liver tissue when exposed to 15 (226%) and 150 mg/l (200%). These results indicate that iron can promote significant alterations in AChE activity which probably is not directly related to the iron content in zebrafish tissues.     

Identification of non-alkaloid acetylcholinesterase inhibitors from Ferulago campestris (Besser) Grecescu (Apiaceae)

Inhibition of Acetylcholinesterase (AChE) is still considered as a strategy for the treatment of neurological disorders such as Alzheimer's disease. Many plant derived alkaloids (such as galantamine and rivastigmine) are known for their AChE inhibitory activity. Recently, other classes of natural compounds such as terpenoids, sesquiterpene glycosides and coumarins have been studied as new AChE inhibitors, with the aim to discover less toxic compounds compared to alkaloidal ones. The Ferulago campestris roots dichloromethane extract was used for a bioassay-guided fractionation for the search of AChE inhibitors. Three coumarin derivatives (umbelliprenin 1, coladonin 2 and coladin 3), three daucane ester derivatives (siol anisate 4, ferutinin 5 and 1-acetyl-5-angeloyl lapiferol 6), two phenol derivatives (2-epilaserine 7 and epielmanticine 8) and one polyacetylene (9-epoxyfalcarindiol 9) were isolated by the bioassay-guided approach. Their structures were characterized on the basis of spectral methods (1D and 2D NMR, and MS spectroscopy). All the isolated compounds were able to inhibit the AChE (IC₅₀ 1.2–0.1 mM) although at higher doses if compared to galantamine (6.7 μM) measured in the same conditions. The most active compounds were the daucane derivative siol anisate 4 and the epielmanticine 8, with IC₅₀ of 0.172 and 0.175 mM respectively.