Frog Blood Research Articles

A METHOD FOR ISOLATING AND SEQUENCING TRYPANOSOME CELLS TO INVESTIGATE SPECIES ASSOCIATIONS IN MULTIPLE MORPHOTYPE INFECTIONS.

Trypanosome infections containing multiple morphologies have been described from all classes of vertebrates, including mammals, birds, non-avian reptiles, amphibians, and fish. These mixed infections make it challenging to evaluate trypanosome diversity, as it is not immediately clear whether the forms present in the bloodstream represent different species or a single pleomorphic species. Amphibians are common hosts for trypanosomes and are often infected by multiple trypanosome morphologies in the bloodstream. Based on morphological observations and life cycle studies, many authors have considered multiple trypanosome morphotypes found infecting the same frogs to be a single pleomorphic species. However, molecular evidence supporting pleomorphic trypanosome species in amphibians is lacking, primarily because linking sequence data to bloodstream trypanosome morphology in mixed infections is extremely challenging. Here we present methods to isolate individual trypanosome cells of 6 morphotypes from frog blood for nested PCR of the 18S rRNA and gGAPDH genes. Single trypanosome cells were isolated by dilution and 3 DNA extraction methods, and 5 nested PCR primer regimes were utilized to optimize amplification from very low starting concentrations. The success rates of extraction methods ranged from 29 to 50% with the use of a Direct PCR kit having the highest success rate. Although the success rate varied in the different combinations of extraction methods and primer regimes, multiple individuals of all 6 trypanosome morphotypes were sequenced for both genes in a novel way that links sequence data to cell morphology by observing isolated cells with a microscope before PCR amplification. All 6 morphologically distinguishable morphotypes coinfecting a frog were genetically distinct. The only other recent molecular study on amphibian trypanosomes also found genetic differences between morphotypes in multiple infections. Together these studies suggest that the occurrence of pleomorphism may be overestimated in amphibian trypanosomes. The methods presented here offer a promising solution to characterize trypanosome diversity within multiple morphotype infections.

Effects of two pesticides on northern leopard frog (Lithobates pipiens) stress metrics: Blood cell profiles and corticosterone concentrations

Amphibians are declining globally. Exposure to pesticides has been implicated in decreasing amphibian immune function, thus increasing their susceptibility to parasites and disease and thereby negatively affecting individuals and populations. Amphibians are likely exposed to neonicotinoids because these widely used insecticides are highly soluble in water and because amphibian freshwater habitats are often embedded in agroecosystems. Herein, we investigate the effects of long-term exposure to two individual neonicotinoids (clothianidin or thiamethoxam) at either low or high concentrations (2.5 or 250 µg/L) on northern leopard frog (Lithobates pipiens) blood cell profiles and concentrations of corticosterone, an energy-mediating hormone associated with stress. Larval frogs from Gosner stage 25 to 46 were exposed to pesticide and control treatments in outdoor mesocosms. Corticosterone concentrations were measured after 6 d of exposure, and blood cell profiles were assessed once frogs reached Gosner stage 46 (following 8 w of exposure). No significant changes were found in erythrocyte counts, leukocyte counts, monocyte to leukocyte ratios or corticosterone concentrations between treatments. However, exposure to either 2.5 or 250 µg/L of clothianidin, or 250 µg/L of thiamethoxam decreased neutrophil to lymphocyte ratios and neutrophil to leukocyte ratios, and exposure to 2.5 µg/L of clothianidin or 250 µg/L of thiamethoxam decreased eosinophil to leukocyte ratios. Our results indicate that long-term exposure to neonicotinoids can alter leukocyte profiles, indicative of a stress response. Future studies should investigate whether chronic exposure to neonicotinoids affect multiple measures of stress differently or influences the susceptibility of amphibians to parasites and pathogens. Our work underscores the importance of continued use of multiple measures of stress for different amphibian species when undertaking ecotoxicological assessments.

Comparison of DNA damage level in irradiated blood cells of mouse, frog and human

Here, we present the comparative studies of DNA damage induced by X-ray irradiation in vitro in mouse, frog and human blood cells and assessed by the “comet test” (alkaline version). We showed that, in contrast to mouse and human cells, DNA repair processes in frog blood cells are not completed by 60 minutes of the post-radiation period. The results suggest that frogs can be used as indicators of environmental radiation pollution. In this regard, additional experiments are needed to assess the different types of external and internal exposure of animals.

The level of DNA damage in mouse hematopoietic cells and in frog and human blood cells, as induced by the action of reactive oxygen species in vitro.

Comparative studies of the level of DNA damage induced in vitro by X-rays (0-8Gy) or hydrogen peroxide (0-300µM) in cells of blood, spleen, and bone marrow of mice and in blood cells of frogs and humans were performed using the alkaline comet assay. For both agents, the levels of induced DNA damage in leucocytes/splenocytes of mice were higher than those in blood cells of frogs and humans, while in human leucocytes, they were comparable with those in frog blood cells. The rate of DNA repair in frog blood cells was very slow. The results suggest that the levels of radiation-induced DNA damage are not in accordance with species radiosensitivity (according to LD50/30) but rather with the intrinsic peculiarities of cells.

Virtual phase conjugation based optical tomography for single-shot three-dimensional imaging.

We propose a virtual phase conjugation (VPC) based optical tomography (VPC-OT) for realizing single-shot optical tomographic imaging systems. Using a computer-based numerical beam propagation, the VPC combines pre-modulation and post-demodulation of the probe beam's wavefront, which provides an optical sectioning capability for resolving the depth coordinates. In VPC-OT, the physical optical microscope system and VPC are coupled using digital holography. Therefore, in contrast to conventional optical tomographic imaging (OTI) systems, this method does not require additional elements such as low-coherence light sources or confocal pinholes. It is challenging to obtain single-shot three-dimensional (3D) tomographic images using a conventional OTI system; however, this can be achieved using VPC-OT, which employs both digital holography and computer based numerical beam propagation. In addition, taking into account that VPC-OT is based on a complex amplitude detection using digital holography, this method allows us to simultaneously obtain quantitative phase contrast images. Using an objective lens with a numerical aperture (NA) of 0.8, we demonstrate a single-shot 3D imaging of frog blood cells with a depth resolution of 0.94 μm.

Reduced innate immunity of Cuban Treefrogs at leading edge of range expansion.

During geographic range expansion, populations of non-indigenous species at the invasion front may benefit from directing resources away from immune defense. To test this hypothesis, we investigated the strength of two innate immune components in populations of invasive Cuban Treefrogs (Osteopilus septentrionalis) in a long-colonized area (core region) and at the invasion front (leading-edge region). First, we compared the region-specific metabolic response of frogs injected with an endotoxin that induces systemic inflammation (lipopolysaccharide, LPS) to sham-injected control frogs pooled from both regions. Males and females were analyzed independently because we detected a sex-related difference in mass-independent metabolism of control frogs, with males exhibiting a significantly higher metabolic rate (F1, 21 =29.02, P<0.001) than females. We observed a significantly higher metabolic rate in LPS-injected core frogs compared with control frogs for both males (P = 0.041) and females (P = 0.007). Conversely, in leading-edge populations, there was no significant difference in the metabolic rate of LPS-injected and control frogs (males, P=0.195; females, P=0.132). Second, we directly compared bacterial killing ability of frog blood plasma between regions. Bactericidal ability of plasma was significantly greater in frogs from the core region in comparison with those at the leading edge (F1, 26 =28.67, P<0.001). For both immune components that we examined, populations from the core exhibited stronger immune responses. Our findings support hypotheses predicting an inverse relationship between immunity and range expansion.

CYTOGENETIC EVALUATION OF THE DRINKING WATER TOXICITY

There was considered the use of biotesting for the assessment of the quality of drinking water from the different water supply sources (artesian, packaged and water-pipe one). The method consists in determination of the toxicants action on the specially selected organisms in the standard conditions with registration of changes at behavior, physiological, cellular and subcellular level using hematological indices of fish, frogs, rats and the lymphocyte cultures of the peripheral human blood. Physical-chemical methods determine only the presence and number of chemical elements in the tested water samples because of the very large number of possible combinations of chemical compounds in water solutions (more than 75 million combinations), including behavior of anthropogenic compounds and the natural vulnerability of the water ecosystems to the combined effects from its toxic influence. As the optimal set of determination of the some structural and functional changes of cell genome as the result of the toxic influence of combination of the chemical compounds in the water solutions was offered the micronuclear test and leukocytic formula of the fish, frog and rat blood as biomarker. The reaction of fish, frog, rat test-organisms on the toxic irritation is presented in the change of qualitative content of the cells of peripheral blood. There were demonstrated the prospects of the use of hematological indices of the following test-organisms: Danio rerio fishes, Xenopus clawed frogs, Wistar rats and also the lymphocytes cultures of the human peripheral blood. The special attention was paid to the assessment of the risk for human health of the toxic substances in drinking water, genotoxicity and cytotoxicity that are revealed using hematological indices of animal cells. The universality of cellular organization opens the wide possibilities for toxicological studies using peripheral blood of the different groups of animals (fish, frogs, rats), human lymphocyte cultures and allow assume the following possibility of extrapolation of the received results on human organism

Quantification of X. laevis vitellogenin by liquid chromatography tandem mass spectrometry

Over the last several decades, there has been an increase in public awareness and regulatory activity in regard to the presence of emerging contaminants in the environment that may have the potential to interact with the endocrine system of exposed wildlife. Alterations in vitellogenin (VTG), a high density yolk precursor protein, can indicate endocrine activity in oviparous species, including many fish and amphibians. While various methodologies and experiments have been performed to characterize baseline VTG concentrations among commonly studied fish species, fewer methodologies for accurately quantifying amphibian VTG are available. Since there is relatively little information available on background VTG levels in male and female frogs, the present investigation set out to quantify baseline levels of VTG in juvenile as well as adult male and female African clawed frogs (Xenopus laevis) using a newly developed liquid chromatography tandem mass spectrometry method. This new methodology for quantifying VTG in X. laevis frog blood plasma can be applied in mechanistic and toxicity studies with X. laevis to better characterize potential endocrine modes of action.

Influence of Hepatozoon parasites on host-seeking and host-choice behaviour of the mosquitoes Culex territans and Culex pipiens

Hepatozoon species are heteroxenous parasites that commonly infect the blood of vertebrates and various organs of arthropods. Despite their ubiquity, little is known about how these parasites affect host phenotype, including whether or not these parasites induce changes in hosts to increase transmission success. The objectives of this research were to investigate influences of the frog blood parasite Hepatozoon clamatae and the snake blood parasite Hepatozoon sipedon on host-seeking and host-choice behaviour of the mosquitoes Culex territans and Culex pipiens, respectively. During development of H. sipedon in C. pipiens, significantly fewer infected mosquitoes fed on uninfected snakes compared to uninfected mosquitoes. When H. sipedon was mature in C. pipiens, the number of infected and uninfected C. pipiens that fed on snakes was not significantly different. Higher numbers of mosquitoes fed on naturally infected snakes and frogs compared to laboratory-reared, uninfected control animals. However, experiments using only laboratory-raised frogs revealed that infection did not significantly affect host choice by C. territans. Behaviour of C. pipiens in the presence of H. sipedon may increase transmission success of the parasite and provide the first evidence of phenotypic changes in the invertebrate host of Hepatozoon parasites.

A comparative study of certain pharmacological effects of lidocaine and ropivacaine

In order to study the safety and efficacy of lidocaine and ropivacaine some of their actions were compared using the parameters namely local anaesthetic effect on rabbit cornea, local anaesthetic effect on the reflex movements of the nostrils of rabbit, analgesic effect on rat, cardiac effect on frog heart and the effect on the blood vessels of frog. The local anaesthetic effect on rabbit cornea and reflex movements of the nostrils of rabbit had longer duration with ropivacaine but there were differences in the onset of action. By rat tail flick method, ropivacaine proved to be more potent and longer acting in its analgesic action. Ropivacaine took longer time to produce bradycardia. There was less number of missed beats of the frog heart with ropivacaine. While lidocaine produced vasoconstriction of frog blood vessels, ropivacaine produced initial vasoconstriction followed by vasodilatation. In all the parameters used, ropivacaine proved to be more potent, longer acting and safer when compared to lidocaine.

Study on Medical Micro-Image Mosaic with SIFT Features

In order to obtain panoramic view of medical micro-image in remote medical diagnosis system, we should mosaic micro-image sequence accurately in remote node. SIFT features are invariant to image scaling, rotation and translation. The aim of this study is to mosaic micro-image sequence by extracting SIFT features. Firstly, search coordinate of potential features through Gaussian pyramid (with 7 octaves × 6 scales) and then filter pseudo-keypoints. Secondly, describe these features with 128-D vector. The next step is to calculate matching keypoint pairs between two time-successive images according to minimum Euclidean distance. In this step, standard deviation comparing is proposed to eliminate wrong matching pairs and appropriate threshold(8000) through experiment is used to insure matching pairs. Then, construct image motion equation considering rotation and translation and compute motion parameter by solving equation according matching pairs. Finally mosaic all images. These steps are applied in 5 micro-image sequences such as slices of lung tissue, spleen, kidney, frog blood cell and sunflower tissue. The experiment results show that the gap between two images is vanishing and the proposed method can satisfy with medical micro-images sequence mosaic.

Experimental Transmission of Hepatozoon clamatae (Apicomplexa: Adeleida) to the Wood Frog, Rana sylvatica, and to the Mosquito Culex pipiens

Hepatozoon clamatae naturally infects the erythrocytes of green frogs (Rana clamitans), bullfrogs (Rana catesbeiana), and northern leopard frogs (Rana pipiens) in northeastern North America and uses the mosquito Culex territans as a definitive host. In this study, we show that the wood frog, Rana sylvatica, supports merogonic development, but not gamogonic development, of this protozoan parasite, and that the mosquito Culex pipiens serves as an experimental definitive host for sporogonic development. Two wood frogs were each force-fed Cx. territans, containing oocysts of H. clamatae in their Malpighian tubules, which had fed on blood of infected green frogs 30 days previously. Free merozoites were observed in 1 wood frog 35 days after inoculation, but intraerythrocytic gamonts were not observed. Fifteen Cx. pipiens were fed on a mixture of infected frog blood and physiological saline. Thirty days after blood feeding, 2 mosquitoes were infected with oocysts of H. clamatae, whereas the other 13 mosquitoes either were negative for infection or had died. The observed absence of gamogonic development of this parasite in wood frogs are discussed in light of previous records of host specificity of Hepatozoon species for their anuran hosts, and the importance of Cx. pipiens as an additional definitive host for H. clamatae.

Plasma membrane electron transport in frog blood vessels

In an attempt to see if frog blood vessels possess a plasma membrane electron transport system, the postcaval vein and aorta isolated from Rana tigrina were tested for their ability to reduce ferricyanide, methylene blue, and 2,6-dichloroindophenol. While the dyes remained unchanged, ferricyanide was reduced to ferrocyanide. This reduction was resistant to inhibition by cyanide and azide. Heptane extraction or formalin fixation of the tissues markedly reduced the capability to reduce ferricyanide. Denuded aortas retained only 30% of the activity of intact tissue. Our results indicate that the amphibian postcaval vein and aorta exhibit plasma membrane electron transport.

Glycation of wood frog ( Rana sylvatica) hemoglobin and blood proteins: In vivo and in vitro studies

The effects of in vivo freezing and glucose cryoprotectant on protein glycation were investigated in the wood frog, Rana sylvatica. Our studies revealed no difference in the fructoselysine content of blood plasma sampled from control, 27 h frozen and 18 h thawed wood frogs. Glycated hemoglobin (GHb) decreased slightly with 48 h freezing exposure and was below control levels after 7 d recovery, while glycated serum albumin was unchanged by 48 h freezing but did increase after 7 d of recovery. In vitro exposure of blood lysates to glucose revealed that the GHb production in wood frogs was similar to that of the rat but was lower than in leopard frogs. We conclude that wood frog hemoglobin was glycated in vitro; however, GHb production was not apparent during freezing and recovery when in vivo glucose is highly elevated. It is possible that wood frog blood proteins have different in vivo susceptibilities to glycation.

A new species of Sycorax Curtis (Diptera, Psychodidae, Sycoracinae) collected on harlequin frogs (Anura: Bufonidae, Atelopus) in the Ecuadorian Andes

Sycorax wampukrum sp. nov. is described from the Amazonian slopes of the Cordillera Oriental of southern Ecuadorian Andes. This new species constitutes the first record of the genus from Ecuador. Males and females of this new species were found in contact with the dorsal surfaces of head, body and extremities of male individuals of harlequin frogs, thus establishing the second record of species of the genus Sycorax feeding on frog blood.

VALIDATION AND NOVEL APPLICATIONS OF THE WHOLE-BLOOD CHEMILUMINESCENCE ASSAY OF INNATE IMMUNE FUNCTION IN WILD VERTEBRATES AND DOMESTIC CHICKENS

The whole-blood chemiluminescence (WBCL) assay is a simple and rapid method of measuring production of reactive oxygen species by circulating leukocytes, particularly heterophils (birds) and neutrophils (other vertebrates). In the interest of substantiating a broadly applicable measure of innate immunity, we investigated the microplate WBCL method for several wildlife species as well as domestic broiler chickens. Lucigenin as a light enhancer was used for all avian blood, wild and domestic, while luminol was used in bear and frog blood. Use of ethylenedinitrilotetraacetic acid (EDTA) as the anticoagulant caused hemolysis of frog blood and decreased WBCL responses in all animals tested. Heparin, even in high concentrations, caused modest to no decrease of WBCL responses. The WBCL response correlated highly with heterophil or neutrophil numbers in all species tested. The WBCL response was tested in freshly collected blood as well as in blood one to five days postcollection in order to determine the utility of this assay for field studies when immediate access to laboratory facilities is not possible. One to three days of delay in performing the test after blood collection caused no, or only a slight, decay of the chemiluminescence response in most animals. Using domestic chickens, we tested the sensitivity of the WBCL method to detect differences between treatment groups and looked for loss of chemiluminescence response over several days using the original blood samples. Significant differences in the WBCL response between experimental groups of broiler chicken were detectable in freshly collected blood, as well as one- to four-day-old blood. Our results show that the innate immune response of populations of wild animals may be successfully compared using this assay, even when blood cannot be tested until a few days after collection.

Avian chromosomes can be examined by injection of erythrocyte nuclei into mouse oocytes.

Avian chromosomes can be examined by injection of erythrocyte nuclei into mouse oocytes.

Linker Histone Subtype Composition and Affinity for Chromatin in Situ in Nucleated Mature Erythrocytes

The replacement linker histones H1(0) and H5 are present in frog and chicken erythrocytes, respectively, and their accumulation coincides with cessation of proliferation and compaction of chromatin. These cells have been analyzed for the affinity of linker histones for chromatin with cytochemical and biochemical methods. Our results show a stronger association between linker histones and chromatin in chicken erythrocyte nuclei than in frog erythrocyte nuclei. Analyses of linker histones from chicken erythrocytes using capillary electrophoresis showed H5 to be the subtype strongest associated with chromatin. The corresponding analyses of frog erythrocyte linker histones using reverse-phase high performance liquid chromatography showed that H1(0) dissociated from chromatin at somewhat higher ionic strength than the three additional subtypes present in frog blood but at lower ionic strength than chicken H5. Which of the two H1(0) variants in frog is expressed in erythrocytes has thus far been unknown. Amino acid sequencing showed that H1(0)-2 is the only H1(0) subtype present in frog erythrocytes and that it is 100% acetylated at its N termini. In conclusion, our results show differences between frog and chicken linker histone affinity for chromatin probably caused by the specific subtype composition present in each cell type. Our data also indicate a lack of correlation between linker histone affinity and chromatin condensation.

Rhythm 'n' biology: what happens if cell biology and techno music meet?

Beauty is in the eye of the beholder. Peering down a microscope, what a layperson may regard as strange fuzzy structures could be an exciting discovery for the trained scientist. On the other hand, while the scientist might only see useful information, the layperson may marvel at the hidden beauty of the microscopic world. To watch bacteria swim towards a food source or an amoeba slowly crawl toward and digest an unfortunate bacterium does hold a certain fascination of watching life taking place at the smallest level. By granting access into these minute worlds, the scanning electron microscope, for instance, has not only allowed scientists to study the surfaces of many cells and organisms at high magnifications, but has also produced many of the images that have graced the covers of or have been featured as full‐page illustrations in non‐scientific magazines. > The colourful patterns from immunofluorescence or confocal laser scanning microscopy also have a striking beauty that goes beyond the scientific content Similarly, fluorescently tagged antibodies and the green fluorescent protein (GFP) have revolutionised light microscopy, and many biological publications today contain familiar images of brightly labelled intracellular structures. And it does not stop at simple pictures; conferences on microbiology or cell biology feature an increasing number of presentations that rely on movies of cellular events. But the colourful patterns produced by immunofluorescence or confocal laser scanning microscopy also have a striking beauty that goes beyond the scientific content they represent. This awareness led some scientists to develop a new concept of depicting their work: a form of multimedia presentation that relates a biological story through text, animation, sound, video and even music such that the speaker is redundant. This approach was demonstrated to the auditorium of the Cinema of the Cell session at this year's ELSO conference, organised by …

Experimental infections of Rana esculenta with Aeromonas hydrophila: a molecular mechanism for the control of the normal flora.

Frogs can be useful models for studying the mechanisms that may regulate their natural microbial flora. Their skin glands produce a secretion containing 20-30 different peptides, some antimicrobial some neurotrophic. As they often live in soil or silt that is rich in microbes, they can be expected to be able to prevent or eliminate infections in very short periods of time. The bacterium Aeromonas hydrophila is widely distributed in nature and is considered as part of the natural flora of frogs and many animals, including humans. From an alternative frog strain of A. hydrophila, Bo-3, we isolated a spontaneous and stable mutant (Bo-3N), resistant to nalidixic acid, here used to follow the host-microbe interactions in experimental infection of mouth and skin of Rana esculenta. The skin peptides had been previously isolated, sequenced and cloned. We showed that skin treatment with a glucocorticoid (GC) cream blocked de novo synthesis of these peptides and, simultaneously, prepropeptide mRNAs disappeared while IkappaBalpha was up-regulated. Experimental mouth infections with 20 million cells of A. hydrophila Bo-3N showed that a normal wild frog can eliminate the bacteria from the mouth within 15 min, while a frog pretreated with GC cream for 1 h could not reduce Bo-3N below 3500 colony-forming units (CFU)/5 microl 'saliva'. An in vitro comparison showed that frog blood or serum allowed bacteria to grow, while the skin secretion killed the bacteria within 10 min. Using different enzyme-linked immunosorbent assays (ELISAs) with rabbit anti-Bo-3 serum as a positive control, we were able to rule out immunoglobulin G (IgG) binding to A. hydrophila. An assay for immunoglobulin M (IgM) (or some other serum component) in frog serum showed binding to A. hydrophila only corresponding to a few per cent of the positive control. For skin infections we bathed the frogs for 10 min in an overnight culture of Bo-3N diluted to about 10(7) CFU/ml. Electrical stimulation after the bath showed, for the total secretion, a two to fourfold increase in the antibacterial activity, while a pretreatment with GC cream reduced the activity to about one-third of that of the non-bathed control frog. HPLC analysis of the peptide pattern confirmed these findings. The survival value of antimicrobial peptides have earlier been demonstrated in vivo and in vitro only in Drosophila. The present experiments are the first combined in vivo and in vitro demonstrations of the function of peptide antibiotics in a vertebrate. One such function is involved in the control of the natural flora.