Ferric Reducing Antioxidant Power and Square Wave Voltammetry for Assay of Low Molecular Weight Antioxidants in Blood Plasma: Performance and Comparison of Methods

The purpose of the present study was to employ two methods—square wave voltammetry (SWV) performed on screen printed sensors and ferric reducing antioxidant power (FRAP)—as suitable tools for the assay of low-molecular-weight antioxidants (LMWAs). LMWAs were assayed by both methods and the resulting data were statistically compared. Plasma samples from five Cinereous vultures accidentally intoxicated with lead were used to represent real biological matrices with different levels of LMWAs. Blood was collected from the birds prior to and one month after treatment with Ca-EDTA. SWV resulted in two peaks. The first peak, with the potential value of 466 ± 15 mV, was recognized as ascorbic and uric acids, while the second one (743 ± 30 mV) represented glutathione, tocopherol, ascorbic acid and in a minor effect by uric acid, too. Contribution of individual antioxidants was recognized by separate assays of LMWA standards. Correlation between peaks 1 and 2 as well as the sum of the two peaks and FRAP was analysed. While peak 1 and the sum of peaks were in close correlation to FRAP results (correlation coefficient of 0.97), the relation between peak 2 and FRAP may be expressed using a correlation coefficient of 0.64. The determination of thiols by the Ellman assay confirmed the accuracy of SWV. Levels of glutathione and other similar structures were stable in the chosen model and it may be concluded that SWV is appropriate for assay of LMWAs in plasma samples. The methods employed in the study were advantageous in minimal sample volume consumption and fast acquisition of results

Elektrokemijsko određivanje metalotioneina kod domaće peradi

Metallothionein (MT) belongs to group of intracellular, low-molecular and cysteine-rich proteins with a molecular weight from 6 to 10 kDa. Owing to their high affinity to heavy metals (Zn, Cd, As, etc.) their main role is homeostatic control and detoxification of metal ions in an organism. In the present work we aimed at suggesting and utilizing electroanalytical techniques to determine content of MT in the blood serum of domestic fowls. Electrochemical measurements were performed with an AUTOLAB Analyser connected to VA-Stand 663, using a standard cell with three electrodes. Particularly, MT was detected by adsorptive transfer stripping technique in connection with differential pulse voltammetry. The detection limit of MT was estimated down to 100 fM (standards only) or down to 100 pM measured in the presence of blood serum. The average content of MT was 21.3 µM. The MT level in hens was about 25 % higher than in cocks. This phenomenon can be related to higher demands on the content of this protein in hens due the requirement for ion transport to form eggshell.Metalotionein (MT) pripada grupi intracelularnih proteina male molekularne mase bogatih cisteinom, s molekularnom masom od 6 do 10 kDa. Zbog njihovog afiniteta prema teškim metalima (Zn, Cd, As, itd.) njihova glavna uloga je homeostatska kontrola i detoksifikacija iona metala u organizmu. U ovom radu predlažu se elektroanalitičke tehnike za određivanje sadržaja MT u krvnom serumu domaće peradi. Elektrokemijska mjerenja izvršena su uređajem AUTOLAB Analyser povezanim s VA-Stand 663, koristeći standardnu ćeliju s tri elektrode. Osim toga MT je određivan tehnikom adsorptivnog transfera, povezanoj s voltmetrijom diferencijalnog pulsa. Granica detekcije MT je procjenjivana do 100 fM (samo standardi) ili do 100 pM, mjereno u prisutnosti krvnog seruma. Prosječni sadržaj MT bio je 21.3 µM. Razina MT kod kokoši bila je otprilike 25% viša nego kod pijetlova. Ta pojava može se objasniti većom potrebom za ovim proteinom kod kokoši zbog transporta iona prilikom stvaranja ljuske jajeta

Biochemical responses and oxidative stress in Francisella tularensis infection: a European brown hare model

<p>Abstract</p> <p>Background</p> <p>The aim of the present study was to investigate biochemical and oxidative stress responses to experimental <it>F. tularensis </it>infection in European brown hares, an important source of human tularemia infections.</p> <p>Methods</p> <p>For these purposes we compared the development of an array of biochemical parameters measured in blood plasma using standard procedures of dry chemistry as well as electrochemical devices following a subcutaneous infection with a wild <it>Francisella tularensis </it>subsp. <it>holarctica </it>strain (a single dose of 2.6 × 10⁹CFU <it>pro toto</it>).</p> <p>Results</p> <p>Subcutaneous inoculation of a single dose with 2.6 × 10⁹colony forming units of a wild <it>F. tularensis </it>strain <it>pro toto </it>resulted in the death of two out of five hares. Plasma chemistry profiles were examined on days 2 to 35 post-infection. When compared to controls, the total protein, urea, lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase were increased, while albumin, glucose and amylase were decreased. Both uric and ascorbic acids and glutathione dropped on day 2 and then increased significantly on days 6 to 12 and 6 to 14 post-inoculation, respectively. There was a two-fold increase in lipid peroxidation on days 4 to 8 post-inoculation.</p> <p>Conclusions</p> <p>Contrary to all expectations, the present study demonstrates that the European brown hare shows relatively low susceptibility to tularemia. Therefore, the circumstances of tularemia in hares under natural conditions should be further studied.</p

Nephrocalcinosis in farmed salmonids: diagnostic challenges associated with low performance and sporadic mortality

Disease conditions that involve multiple predisposing or contributing factors, or manifest as low performance and/or low-level mortality, can pose a diagnostic challenge that requires an interdisciplinary approach. Reaching a diagnosis may also be limited by a lack of available clinical profile parameter reference ranges to discriminate healthy fish from those affected by specific disease conditions. Here, we describe our experience investigating poorly performing rainbow trout (Oncorhynchus mykiss) in an intensive recirculation aquaculture, where reaching a final diagnosis of nephrocalcinosis was not as straightforward as one would wish. To list the issues making the diagnosis difficult, it was necessary to consider the creeping onset of the problem. Further diagnostic steps needed to ensure success included obtaining comparative data for fish blood profiles and water quality from both test and control aquacultural systems, excluding infections with salmonid pathogenic agents and evaluating necropsy findings. Major events in the pathophysiology of nephrocalcinosis could be reconstructed as follows: aquatic environment hyperoxia and hypercapnia → blood hypercapnia → blood acid-base perturbation (respiratory acidosis) → metabolic compensation (blood bicarbonate elevation and kidney phosphate excretion) → a rise in blood pH → calcium phosphate precipitation and deposition in tissues. This case highlights the need to consider the interplay between water quality and fish health when diagnosing fish diseases and reaching causal diagnoses

Mycoplasma gallisepticum infection in the grey partridge Perdix perdix: outbreak description, histopathology, biochemistry and antioxidant parameters

<p>Abstract</p> <p>Background</p> <p>The grey partridge is an important game bird in Europe that has declined considerably over the last decades. The production and release of farm-bred birds can be threatened by infectious agents. The objective of this study was to describe the outbreak, pathology, and blood and tissue biochemical responses in a flock of grey partridges naturally infected with <it>Mycoplasma gallisepticum</it>.</p> <p>Results</p> <p>Morbidity and mortality rates were 100% and 60%, respectively. Necropsy revealed an accumulation of caseous exudate within the infraorbital sinuses, tracheitis, pneumonia and airsacculitis. There were significant increases in activities of lactate dehydrogenase, creatine kinase and amylase, and levels of total protein and glucose in <it>Mycoplasma</it>-infected birds when compared to control. Catalase showed significantly lower activity in the heart, lungs, liver and gonads of <it>Mycoplasma</it>-infected birds. Glutathione-S-transferase activity was elevated in the eye and the associated infraorbital sinus and kidneys, and decreased in the liver. Decreased levels of reduced glutathione were found in the heart, kidneys, liver and gonads. The activity of glutathione reductase was lower only in the lungs. Compared to healthy birds, mycoplasmosis in the grey partridge caused significant differences in the level of lipid peroxidation in lungs and plasma (p < 0.05), while the ferric reducing antioxidant power was lower in the heart and kidneys (p < 0.01). Significant correlations among responses of the antioxidant parameters were found namely in the heart, lungs, spleen, liver and plasma. There were also numerous significant inter-tissue correlations of all the studied antioxidant parameters.</p> <p>Conclusions</p> <p>The present study demonstrates the high susceptibility of grey partridges to natural infection by <it>M. gallisepticum</it>, the severity of the disease based on histopathology, and the modulation of blood chemical profiles and oxidative stress-associated parameters in the avian hosts, thus enhancing the understanding of the pathogenesis of mycoplasmosis in birds. Moreover, the reported reference values can be useful for the evaluation of the state of health in grey partridges.</p

Shapes of Differential Pulse Voltammograms and Level of Metallothionein at Different Animal Species

Metallothioneins play a key role in maintaining homeostasis of essential metals and in protecting of cells against metal toxicity as well as oxidative damaging. Excepting humans, blood levels of metallothionein have not yet been reported from any animal species. Blood plasma samples of 9 animal species were analysed by the adsorptive transfer stripping technique to obtain species specific voltammograms.Metallothioneins play a key role in maintaining homeostasis of essential metals and in protecting of cells against metal toxicity as well as oxidative damaging. Excepting humans, blood levels of metallothionein have not yet been reported from any animal species. Blood plasma samples of 9 animal species were analysed by the adsorptive transfer stripping technique to obtain species specific voltammograms

Comparison of diagnostic methods for Tetracapsuloides bryosalmonae detection in salmonid fish.

Diagnostic accuracy of pathogen detection depends upon the selection of suitable tests. Problems can arise when the selected diagnostic test gives false-positive or false-negative results, which can affect control measures, with consequences for the population health. The aim of this study was to compare sensitivity of different diagnostic methods IHC, PCR and qPCR detecting Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonid fish and as a consequence differences in disease prevalence. We analysed tissue from 388 salmonid specimens sampled from a recirculating system and rivers in the Czech Republic. Overall prevalence of T. bryosalmonae was extremely high at 92.0%, based on positive results of at least one of the above-mentioned screening methods. IHC resulted in a much lower detection rate (30.2%) than both PCR methods (qPCR32: 65.4%, PCR: 81.9%). While qPCR32 produced a good match with IHC (60.8%), all other methods differed significantly (p < .001) in the proportion of samples determined positive. Both PCR methods showed similar sensitivity, though specificity (i.e., the proportion of non-diseased fish classified correctly) differed significantly (p < .05). Sample preservation method significantly (p < .05) influenced the results of PCR, with a much lower DNA yield extracted from paraffin-embedded samples. Use of different methods that differ in diagnostic sensitivity and specificity resulted in random and systematic diagnosis errors, illustrating the importance of interpreting the results of each method carefully

Uporaba voltmetrije za određivanje tiola i metalotioneina male molekularne mase u krvi svinje (Sus scrofa domestica)

Metallothioneins (MT) play a key role in maintaining the homeostasis of essential metals and in protecting of cells against metal toxicity as well as cell oxidative damaging. The aim of this work is to propose a new approach for processing a biological sample for analysis of thiols including metallothioneins. Moreover, the proposed procedure is tested on quantification of MT and total thiol content in blood serum of pig (Sus scrofa domestica), which has not been previously been performed. The blood serum (10 ml) was collected and transferred to 0.2 M phosphate buffer (990 ml). The sample (100 × diluted) was placed in a thermomixer, where heat denaturation of most of the proteins proceeded. The processed blood serum sample was electrochemically measured to determine total content of thiols (cysteine, glutathione, metallothionein and other low molecular thermostable thiols) and content of MT. The average level of the thiols and MT were estimated as 165 ± 20 mM and 5.2 ± 0.6 mM, respectively.Metalotioneini (MT) igraju ključnu ulogu u održavanju homeostaze esencijalnih metala i u zaštiti od toksičnih metala te od oštećivanja stanice oksidacijom. Cilj ovog rada je predložiti novi pristup obradi bioloških uzoraka za analizu tiola, uključujući metalotioneine. Osim toga, predloženi postupak se testira pri kvantifikaciji MT i ukupnog sadržaja tiola u krvnom serumu svinje (Sus scrofa domestica), što se dosad nije radilo. Prikupljan je krvni serum (10 ml) te prebacivan u 0.2 M fosfatni pufer (990 ml). Uzorak (100 × razrijeđen) je stavljen u termomikser gdje se nastavila toplinska denaturacija većine proteina. Obrađeni uzorak seruma je elektrokemijski izmjeren da bi se odredio ukupni sadržaj tiola (cistein, glutation, metalotionein i drugi termostabilni tioli male molekularne mase) te sadržaj MT. Prosječna razina tiola i MT su procijenjeni na 165 ± 20 mM, odnosno 5.2 ± 0.6 mM

of the Moravian Karst Protected Landscape Area

Abstract Host traits and phylogeny can determine infection risk by driving pathogen transmission and its ability to infect new hosts. Predicting such risks is critical when designing disease mitigation strategies, and especially as regards wildlife, where intensive management is often advocated or prevented by economic and/or practical reasons. We investigated Pseudogymnoascus [Geomyces] destructans infection, the cause of white-nose syndrome (WNS), in relation to chiropteran ecology, behaviour and phylogenetics. While this fungus has caused devastating declines in North American bat populations, there have been no apparent population changes attributable to the disease in Europe. We screened 276 bats of 15 species from hibernacula in the Czech Republic over 2012 and 2013, and provided histopathological evidence for 11 European species positive for WNS. With the exception of Myotis myotis, the other ten species are all new reports for WNS in Europe. Of these, M. emarginatus, Eptesicus nilssonii, Rhinolophus hipposideros, Barbastella barbastellus and Plecotus auritus are new to the list of P. destructans-infected bat species. While the infected species are all statistically phylogenetically related, WNS affects bats from two suborders. These are ecologically diverse and adopt a wide range of hibernating strategies. Occurrence of WNS in distantly related bat species with diverse ecology suggests that the pathogen may be a generalist and that all bats hibernating within the distribution range of P. destructans may be at risk of infection

Case report: Filarial infection of a parti-coloured bat: Litomosa sp. adult worms in abdominal cavity and microfilariae in bat semen

BackgroundFilarial infections have been understudied in bats. Likewise, little is known about pathogens associated with the reproductive system in chiropterans. While semen quality is critical for reproductive success, semen-borne pathogens may contribute to reproductive failure.MethodsFor the first time we performed electroejaculation and used computer-assisted semen analysis to provide baseline data on semen quality in a parti-coloured bat (Vespertilio murinus).ResultsThe semen quality values measured in the V. murinus male appeared high (semen concentration = 305.4 × 106/mL; progressive and motile sperm = 46.58 and 60.27%, respectively). As an incidental finding, however, microfilariae were observed in the bat semen examined. At necropsy, eight adult filarial worms, later genetically identified as Litomosa sp., were found in the peritoneal cavity, close to the stomach, of the same particoloured bat male dying as a result of dysmicrobia and haemorrhagic gastroenteritis in a wildlife rescue centre. Histopathology revealed microfilariae in the testicular connective tissue and the epidydimal connective and fat tissues. A PCR assay targeting cytochrome c oxidase subunit 1 confirmed that adult worms from the peritoneal cavity and testicular microfilariae were of the same filarial species. Mildly engorged argasid mite larvae attached to the bat skin proved negative for filarial DNA and the adult filarial worms proved negative for endosymbiont Wolbachia.ConclusionWhile the standard filarial life cycle pattern involves a vertebrate definitive host and an invertebrate vector, represented by a blood-sucking ectoparasite, our finding suggests that microfilariae of this nematode species may also be semen-borne, with transmission intensity promoted by the polygynous mating system of vespertilionid bats in which an infected male mates with many females during the autumn swarming. Presence of microfilariae may be expected to decrease semen quality and transmission via this route may challenge the success of reproductive events in females after mating. Further investigation will be necessary to better understand the bat-parasite interaction and the life cycle of this filarial worm