Perkinsus infection is associated with alterations in the level of global DNA methylation of gills and gastrointestinal tract of the oyster Crassostrea gasar.

Bivalves are filter feeders that obtain food from seawater that may contain infectious agents, such as the protozoan parasites Perkinsus marinus and P. olseni that are associated with massive mortalities responsible for losses in the aquaculture industry. Despite all physical and chemical barriers, microorganisms cross epithelia and infect host tissues to cause pathologies. Epigenetics mechanisms play important roles in a variety of human processes, from embryonic development to cell differentiation and growth. It is currently emerging as crucial mechanism involved in modulation of host-parasite interactions and pathogenesis, promoting discovery of targets for drug treatment. In bivalves, little is known about epigenetic mechanism in host parasite interactions. The objective of the present study was to evaluate the effect of Perkinsus sp. infections on DNA methylation levels in tissues of Crassostrea gasar oysters. Samples were collected in 2015 and 2016 in the Mamanguape River estuary (PB). Oyster gills were removed and used for Perkinsus sp. DIAGNOSIS: Gills (G) and gastrointestinal tract (GT), as well as cultured P. marinus trophozoites were preserved in 95% ethanol for DNA extractions. DNA methylation levels were estimated from G and GT tissues of uninfected (n=60) and infected oysters (n=60), and from P. marinus trophozoites, by ELISA assays. Results showed that the mean prevalence of Perkinsus sp. infections was high (87.3%) in 2015 and moderate (59.6%) in 2016. DNA methylation levels of G and GT tissues were significantly lower in infected oyster than in uninfected oysters, suggesting that infections are associated with hypomethylation. Methylation level was significantly higher in G than in GT tissues, indicating a likely tissue-specific mechanism. P. marinus trophozoites showed 33% methylation. This was the first study that confirms alterations of DNA methylation in two tissues of C. gasar oysters in association with Perkinsus sp. infections.

The 60S ribosomal protein L13 is the most preferable reference gene to investigate gene expression in selected organs from turkeys and chickens, in context of different infection models.

Evaluation of reference genes for expression studies in chickens and turkeys is very much limited and unavailable for various infectious models. In this study, eight candidate reference genes HMBS, HPRT1, TBP, VIM, TFRC, RPLP0, RPL13 and RPS7 were evaluated by five different algorithms (GeNorm, NormFinder, BestKeeper©, delta CT, RefFinder) to assess their stability. In order to analyze a broad variation of tissues, spleen, liver, caecum and caecal tonsil of different aged specific pathogen free (SPF) layer chickens and commercial turkeys, uninfected or infected with the extracellular pathogen Histomonas meleagridis, were included. For tissue samples from SPF chickens RPL13 and TBP were found to be the most stable reference genes. Further testing of RPL13 and TBP in the same organs of uninfected and infected SPF broiler chickens with the intracellular pathogen fowl aviadenovirus confirmed this finding. In tissue samples from turkeys, a stable expression of RPL13 and TFRC genes was noticed. Overall, the determined reference genes should be considered whenever gene expression studies in spleen, liver, caecum and caecal tonsil of chickens and turkeys are performed.

Comparison of haemocytic parameters among flat oyster Ostrea edulis stocks with different susceptibility to bonamiosis and the Pacific oyster Crassostrea gigas.

Farming of the flat oyster Ostrea edulis in Europe is severely constrained by the protozoan Bonamia ostreae. The introduction of the resistant species Crassostrea gigas has been a relief for the farmers, while the pilot programmes to select O. edulis strains resistant to bonamiosis performed in various countries can be seen as a promising strategy to minimise the effects of bonamiosis. However, the physiological bases of this differential susceptibility remain unknown. A search for an explanation of the intra and interspecific differences in oyster susceptibility to bonamiosis was accomplished by comparing some immune parameters among various O. edulis stocks and C. gigas. On December 2003, naïve and Bonamia-relatively resistant flat oysters from Ireland, Galician flat oysters and Pacific oysters C. gigas were deployed in a Galician area affected by bonamiosis; haemolymph samples were taken in February and May 2004. A new oyster deployment at the same place was carried out on June 2004 and haemolymph sampling was performed on April 2005. On November 2004, new sets of Irish flat oysters and C. gigas were deployed in Ireland and haemolymph sampling was performed in June 2005. Various haemocytic parameters were measured: total and differential haemocyte count, phagocytic ability, respiratory burst (superoxide anion [O(2)(-)] and hydrogen peroxide [H(2)O(2)]) and nitric oxide [NO] production. The comparison of the parameters was carried out at 3 levels: (1) between O. edulis and C. gigas, (2) among O. edulis stocks with different susceptibility to bonamiosis, and (3) between Bonamia-infected and non infected O. edulis. In addition, haemocyte-B. ostreaein vitro encounters were performed to analyse interspecific differences in the haemocytic respiratory burst, using flow cytometry. Significant differences associated with total and differential haemocyte count, and respiratory burst between O. edulis and C. gigas were detected, which could be linked to differences in susceptibility to bonamiosis between both species. Additionally, significant changes in total and differential haemocyte count, and respiratory burst of O. edulis associated with B. ostreae infection were found. However, no consistent difference in any haemocyte parameter between the O. edulis stocks involved in the study was recorded.

The origin of malignant malaria.

Plasmodium falciparum, the causative agent of malignant malaria, is among the most severe human infectious diseases. The closest known relative of P. falciparum is a chimpanzee parasite, Plasmodium reichenowi, of which one single isolate was previously known. The co-speciation hypothesis suggests that both parasites evolved separately from a common ancestor over the last 5-7 million years, in parallel with the divergence of their hosts, the hominin and chimpanzee lineages. Genetic analysis of eight new isolates of P. reichenowi, from wild and wild-born captive chimpanzees in Cameroon and Côte d'Ivoire, shows that P. reichenowi is a geographically widespread and genetically diverse chimpanzee parasite. The genetic lineage comprising the totality of global P. falciparum is fully included within the much broader genetic diversity of P. reichenowi. This finding is inconsistent with the co-speciation hypothesis. Phylogenetic analysis indicates that all extant P. falciparum populations originated from P. reichenowi, likely by a single host transfer, which may have occurred as early as 2-3 million years ago, or as recently as 10,000 years ago. The evolutionary history of this relationship may be explained by two critical genetic mutations. First, inactivation of the CMAH gene in the human lineage rendered human ancestors unable to generate the sialic acid Neu5Gc from its precursor Neu5Ac, and likely made humans resistant to P. reichenowi. More recently, mutations in the dominant invasion receptor EBA 175 in the P. falciparum lineage provided the parasite with preference for the overabundant Neu5Ac precursor, accounting for its extreme human pathogenicity.

Variations in the carbohydrate expression pattern and in lesions of the uterine horns of BALB/c mice infected with different Tritrichomonas foetus isolates.

Bovine tritrichomonosis, a sexually transmitted disease caused by the protozoan Tritrichomonas foetus, is characterized by producing reproductive alterations in cattle. Carbohydrates on the surface of the uterine epithelium are involved in the process of adhesion and colonization of the protozoan. The murine model has proved to be an inexpensive, practical and representative alternative to study the lesions produced in the natural host. For this work, during the first stage, 6-8 week old female BALB/c mice were inoculated with 24 different T. foetus isolates in order to classify them according to their pathogenicity. Then, seven isolates were selected and processed with lectin histochemistry to determine if the differences in pathogenicity corresponded to the changes found in the uterine carbohydrate expression pattern. In this work, we demonstrate the differences in the expression of the carbohydrate pattern between infected and uninfected mice. In addition, within the group of infected mice, differences were found in the degree of pathogenicity of the isolates, thus evidencing their biological variability.

Retortamonads from vertebrate hosts share features of anaerobic metabolism and pre-adaptations to parasitism with diplomonads.

Although the mitochondria of extant eukaryotes share a single origin, functionally these organelles diversified to a great extent, reflecting lifestyles of the organisms that host them. In anaerobic protists of the group Metamonada, mitochondria are present in reduced forms (also termed hydrogenosomes or mitosomes) and a complete loss of mitochondrion in Monocercomonoides exilis (Metamonada:Preaxostyla) has also been reported. Within metamonads, retortamonads from the gastrointestinal tract of vertebrates form a sister group to parasitic diplomonads (e.g. Giardia and Spironucleus) and have also been hypothesized to completely lack mitochondria. We obtained transcriptomic data from Retortamonas dobelli and R. caviae and searched for enzymes of the core metabolism as well as mitochondrion- and parasitism-related proteins. Our results indicate that retortamonads have a streamlined metabolism lacking pathways for metabolites they are probably capable of obtaining from prey bacteria or their environment, reminiscent of the biochemical arrangement in other metamonads. Retortamonads were surprisingly found do encode homologs of components of Giardia's remarkable ventral disk, as well as homologs of regulatory NEK kinases and secreted lytic enzymes known for involvement in host colonization by Giardia. These can be considered pre-adaptations of these intestinal microorganisms to parasitism. Furthermore, we found traces of the mitochondrial metabolism represented by iron­sulfur cluster assembly subunits, subunits of mitochondrial translocation and chaperone machinery and, importantly, [FeFe]­hydrogenases and hydrogenase maturases (HydE, HydF and HydG). Altogether, our results strongly suggest that a remnant mitochondrion is still present.

Expression and localization of MCsialec, a sialic acid-specific lectin in the marine bivalve Manila clam, Ruditapes philppinarum.

A novel sialic acid-specific lectin (MCsialec) was detected from an expressed sequenced tag (EST) sequence from Manila clam haemocytes infected with Perkinsus olseni. The cDNA of the lectin was cloned using gene-specific primers based on a previously determined EST and characterized. The full-length cDNA of MCsialec is 603 bp in length and encodes a polypeptide of 200 amino acids with a calculated molecular mass of 21.928 kDa. Sequence alignment and protein motif analyses showed that MCsialec shares identity with sialic acid-specific invertebrate lectins from Cepaea hortensis, Helix pomatia and Haliotis discus discus. The lectin was expressed in Escherichia coli M15 cells and purified using a Ni-NTA His-binding resin matrix for antibody production. The presence of the lectin in various tissues of Perkinsus-infected and uninfected Manila clams was analysed by both PCR and immunohistochemical localization assays. MCsialec was detected in each tissue of the clams; however, upon infection, the level of expression of the lectin increased in each tissue. Vibrio tapetis infection also induced high-level expression of MCsialec in the haemocytes. These data suggest that MCsialec plays a crucial role in the immune system of the Manila clam during pathogenic infection.

Response of Mytilus galloprovincialis (L.) to increasing seawater temperature and to marteliosis: metabolic and physiological parameters.

In the context of climate change the present work aimed to illustrate whether the energetic and metabolic pattern of mussels Mytilus galloprovincialis will be affected by increase in the temperature of seawater. Moreover we examined whether an outbreak of Marteilia sp. infestation as a result of increase in sea water temperature will impair the energetic balance of mussels. M. galloprovincialis was acclimated at 18 degrees C, 24 degrees C, 26 degrees C and 28 degrees C for 30 days and the energetic pattern of its tissues was estimated by determining the factor Scope for Growth (SFG), while the metabolic pattern of mussels was estimated by determining the activities of pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK). The decrease in PK activity and the decrease in the ratio PK/PEPCK indicated an activation of anaerobic component of metabolism during acclimation of mussels at temperature 24 degrees C. At temperatures higher than 24 degrees C the values of SFG turned negative probably associated with a significant reduction in clearance rate. Compared to the non infected mussels, the SFG values of infected mussels were significantly lower (P<0.05). These differences were attributed to the higher filtration rate and the lower absorption efficiency detected in the infected mussels. Also the degree of SFG reduction is dependent on the intensity levels of infection by Marteilia sp.

Heat shock protein (hsp70) expression and thermal tolerance in sublethally heat-shocked eastern oysters Crassostrea virginica infected with the parasite Perkinsus marinus.

To investigate whether sublethal heat shock protects Perkinsus marinus (Dermo)-infected oysters Crassostrea virginica from lethal heat stress, and the effects of P. marinus infection on sublethal heat shock response, oysters were first experimentally challenged with P. marinus. Then, when infections in oysters progressed to moderate levels (parasite burden = 10(4) to 10(5) cells g(-1) wet tissue weight), oysters were treated with a sublethal heat shock at 40 degrees C for 1 h (heat shock + Dermo challenge). Other treatment groups included heat-shocked, unchallenged (non-P. marinus challenged) oysters and non-heat-shocked, P. marinus-challenged and -unchallenged oysters. Thermal tolerance was compared among these treatments by administering a lethal heat treatment at 44 degrees C for 1 h, 7 d after sublethal heat shock. Sublethal heat shock enhanced survival to lethal heat treatment in both P. marinus-challenged and -unchallenged oysters. Although levels of hsp70 isoforms (hsp69 and hsp72) did not vary significantly by heat shock or infection with P. marinus, responses due to these treatments were apparent when comparing hsp70 levels within infected and uninfected oysters. Infection enhanced expression of hsp69, regardless of whether oysters were heat shocked or not. In uninfected oysters, hsp72 increased due to heat shock 2 and 7 d post heat shock. Overall, this study demonstrates that heat shock can improve survival in oysters, even in oysters infected with P. marinus. Expression of hsp70 varied among isoforms after sublethal and lethal heat shocks and in infected and uninfected oysters. The heat shock response was not negatively affected by P. marinus infection.

Hormone levels and infection of Haemoproteus danilewskyi in free-ranging blue jays (Cyanocitta cristata).

Annual spring relapse of blood parasite infections in birds is believed to be the result of hormonal changes associated with breeding. As part of a larger study on the epizootiology of Haemoproteus danilewskyi in blue jays in south-central Florida, we studied the relationship between H. danilewskyi infections and levels of luteinizing hormone, prolactin, progesterone, testosterone, estradiol, and corticosterone. We found a positive association between intensity of H. danilewskyi infection and corticosterone levels in females but not in males. We also found no association between infection and levels of prolactin, luteinizing hormone, progesterone, testosterone, or estradiol in males or females. In addition, we found a positive relationship between levels of corticosterone and handling time and between corticosterone and testosterone levels. We suggest a possible influence of corticosterone on spring relapse of Haemoproteus spp. infections in birds but provide no support for the influence of breeding hormones on relapse of these parasites.

Cytokine gene signatures in neural tissue of horses with equine protozoal myeloencephalitis or equine herpes type 1 myeloencephalopathy.

This study was designed to determine the relative levels of gene transcription of selected pathogens and cytokines in the brain and spinal cord of 12 horses with equine protozoal myeloencephalitis (EPM), 11 with equine herpesvirus type 1 (EHV-1) myeloencephalopathy, and 12 healthy control horses by applying a real time pcr to the formalin-fixed and paraffin-embedded tissues. Total rna was extracted from each tissue, transcribed to complementary dna (cDNA) and assayed for Sarcocystis neurona, Neospora hughesi, EHV-1, equine GAPDH (housekeeping gene), tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10 AND IL-12 p40. S neurona cdna was detected in the neural tissue from all 12 horses with EPM, and two of them also had amplifiable cDNA of N hughesi. The relative levels of transcription of protozoal cdna ranged from 1 to 461 times baseline (mean 123). All the horses with ehv-1 myeloencephalopathy had positive viral signals by PCR with relative levels of transcription ranging from 1 to 1618 times baseline (mean 275). All the control horses tested negative for S neurona, N hughesi and EHV-1 cdna. The cytokine profiles of each disease indicated a balance between pro- and anti-inflammatory markers. In the horses with epm the pro-inflammatory Th1 cytokines (IL-8, TNF-alpha and IFN-gamma) were commonly expressed but the anti-inflammatory Th2 cytokines (IL-4, IL-6 AND IL-10) were absent or rare. In the horses with ehv-1 the proinflammatory cytokine IL-8 was commonly expressed, but IL-10 and IFN-gamma were not, and TNF-alpha was rare. Tissue from the control horses expressed only the gene GAPDH.

[Treatment of Tritrichomonas foetus in a cat colony with delayed release ronidazole tablets in the small intestine]. / Behandlung einer an Tritrichomonas foetus erkrankten Katzen-Kolonie mittels Ronidazoltabletten unter verzögerter Wirkstofffreisetzung im Dünndarm.

Seven abyssinian cats (two male, five female) showed intermittent green-yellow mucous diarrhoea, sometimes an inflammation of the anal region and faecal incontinence even after long-time treatment with fenbendazole against Giardia. During necropsy of one of the cats, which had to be euthanized due to another disease, the gut wall of small and large intestine appeared macroscopically thickened. Histological examination indicated flagellates in the lumen of the intestine (initiating at the jejunum) and in the crypts. However Giardia could be excluded. in this case. By PCR of the faeces Tritrichomonas (T) foetus was diagnosed in five of six cats of this colony. Five remaining animals (another cat had to be euthanized) were treated with about 30 mg per kg BW ronidazole p. o. (rededication; Ridzol 10% Bt®, Dr. Hesse Tierpharma GmbH & Co. KG, Germany) daily over 14 days. The special gastro-resistant processing of the ronidazole should ensure a targeted effects. Animals were treated consecutively, isolated from the other cats and were daily examined clinically and neurologically. Neurotoxic adverse effects appeared slightly, therefore--as a precaution--the treatment of two cats was paused for one day. After treatment of all cats, T. foetus wasn't diagnosed by PCR over the period of 345 to > 800 days in any cat. One animal had dubious findings in the ninth week after treatment. Hence it was still kept isolated from the group and PCR showed a negative result at all times afterwards. The treatment protocol shows that elimination of problematic protozoal infections is possible in cat colonies.

Molecular Characterization and Biochemical and Histopathological Aspects of the Parasitism of Haemoproteus spp. in Southern Caracaras (Caracara plancus).

Haemoproteid species have a wide global distribution, and they have been described in falcon species in several parts of the world. However, few studies in South America have focused on these birds. Haemoproteus spp. infections have been reported as the causative agents of serious histopathological changes, which can lead to the death of the host. Thus, this study aimed to molecularly and phylogenetically characterize Haemoproteus spp. in Caracara plancus, to characterize aspects of parasitism through clinical analysis and biochemical parameters, and to describe the histopathology of infection. To examine these aspects, 5 southern caracaras were examined clinically, and blood samples were collected. Blood smears were subsequently utilized in parasitemia calculations, PCR amplification, and serum biochemical investigations. Histological sections of the liver, kidneys, spleen, and heart were examined to check for possible histopathological changes. The birds showed clinical signs such as pallor and prostration that are consistent with Haemoproteus spp. infection. Moreover, the examination of the blood smears revealed 0.07% parasitemia in young gametocytes only. The PCR and sequencing results confirmed that the parasites belonged to Haemoproteus spp. The activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzymes, albumin, total serum proteins, and enzymatic urea were first described in C. plancus and serve as reference for future studies of bird species parasitized by Haemoproteus spp. Histopathology results showed signs of injury that were consistent with haemosporidian infection in the tissues of the analyzed organs. The present study is preliminary, and additional studies of Haemoproteus spp. infections in other bird species are needed to better understand the relationship between parasites and hosts, because despite the low parasitemia recorded, biochemical and histopathological changes in various organs were observed.

Trade-off between immunocompetence and growth in magpies: an experimental study.

A trade-off between immunity and growth has repeatedly been suggested, mainly based on laboratory and poultry science, but also from experiments where parasitism intensity was manipulated in field bird populations. However, as resource allocation to different activities (or organs) during growth is difficult to manipulate, this trade-off has only been experimentally tested by studying the effects of non-pathogenic antigens. By providing some nestling magpies (Pica pica) with methionine, a sulphur amino acid that specifically enhances T-cell immune response in chickens, we investigated this trade-off by directly affecting allocation of limited resources during growth. Results were in accordance with the hypothetical trade-off because nestlings fed with methionine showed a lower growth rate during the four days of methionine administration, but a larger response when fledglings were challenged with phytohaemagglutinin (a measure of the intensity of T-lymphocyte-mediated immune responsiveness) than control nestlings. Surprisingly, we found that control and experimental nestlings fledged with similar body mass, size and condition, but experimental nestlings suffered less from blood parasites (Haemoproteus) and had fewer lymphocytes (a widely used measure of health status) than control nestlings, suggesting a negative effect of blood parasites or other pathogens on nestling growth.

Identification of glycosomes and metabolic end products in pathogenic and nonpathogenic strains of Cryptobia salmositica (Kinetoplastida: Bodonidae).

Whole cell lysates of pathogenic and nonpathogenic strains of Cryptobia salmositica were subjected to subcellular fractionation using differential and isopycnic centrifugation in sucrose. The glycolytic enzymes hexokinase, fructose-1,6-biphosphate aldolase, triosephosphate isomerase, glucosephosphate isomerase and glyceraldehyde-3-phosphate-dehydrogenase and the peroxisomal enzyme catalase were associated with a microbody that had a buoyant density in sucrose of 1.21 g cm-3. Lactate dehydrogenase was detected in whole cell lysates, but not in purified organelles. A microbody with a positive reaction for catalase was detected in electron microscope sections of the pathogenic and nonpathogenic strains. These catalase-containing microbodies fused with lipid bodies and vacuoles, arose by division from pre-existing microbodies and expelled their contents into the cytoplasm of the cell. Both strains also modified the catalase content in their microbodies. Under aerobic conditions, they metabolized glucose to pyruvate and lactate. We conclude that part of the glycolytic pathway in C. salmositica is compartmentalized in a microbody called the glycosome.

Lectin binding pattern in the uterus of pregnant mice infected with Tritrichomonas foetus.

Bovine genital tritrichomonosis is caused by the protozoon Tritrichomonas foetus and leads to embryonic death and abortion. The complexity of handling bovine experimental systems has led to the development of alternative models. The infection has been reproduced in pregnant BALB/c mice. In the pathogenesis of the disease, adhesion of the protozoon to host cell surface glycoproteins is important. Labelling with soya bean agglutinin (SBA) and peanut agglutinin (PNA) lectins increases in the luminal and glandular uterine epithelium of non-pregnant infected mice. The aim of the present study was to determine whether these changes also occur in pregnant infected BALB/c mice. Female BALB/c mice were inoculated intravaginally with T. foetus and, 15 ± 3 days post infection, were paired with males overnight. Infected and control mice were sacrificed 6, 8 and 10 days later. Samples of uterus were labelled with a panel of biotinylated lectins. Infected mice showed increased binding of PNA and SBA. There was also increased binding of concanavalin (Con-A) by luminal epithelium and Ricinus communis agglutinin (RCA-1) by glandular epithelium at day 6 post coitum. These changes may be due to the production of enzymes by T. foetus, which could act to enhance adhesion and colonization and thus favour infection.

Serum proteinogram, acute phase proteins and immunoglobulins in dogs experimentally infected with Rangelia vitalii.

The present study aimed to evaluate the serum proteinogram, acute phase proteins (APPs) and immunoglobulins (Igs) of dogs experimentally infected by Rangelia vitalii in the acute phases of the disease. Banked serum samples collected on days 0, 10 and 20 during a previously reported R. vitalii experimental infection were used to analyze the serum proteinogram, APPs (C-reactive protein - CRP and alpha-1-acid glycoprotein - AGP) and Igs (IgM, IgG, IgA and IgE) in the current study. Total protein and albumin level were significantly (P<0.05) decreased at day 10 PI and 20 PI in infected sera compared to the control sera. Alpha-1 globulin (day 10 PI) and gamma globulin (day 20 PI) were increased (P<0.01) in infected sera. Alpha-2 globulin (days 10 and 20 PI) and beta-2 globulin (day 10 PI) were decreased (P<0.05) in infected sera compared to control sera. Beta-1 globulin fraction did not differ statistically between sera. Serum CRP and AGP concentrations were significantly increased (P<0.05) at days 10 and 20 PI in infected sera. IgG was increased at days 10 (P<0.05) and 20 PI (P<0.01) in infected sera. Furthermore, it was also observed an increase (P<0.01) in the levels of IgM, IgA, and IgE in infected sera than control sera. We conclude that R. vitalii infection causes alterations in the proteinogram, and increases in the levels of APPs and Igs. Further studies are essentials to define the causes of these pathological changes in this disease.

Adenosine levels in serum and adenosine deaminase activity in blood cells of dogs infected by Rangelia vitalii.

Ecto-adenosinedeaminase (E-ADA) plays an important role in the production and differentiation of blood cells as well as in the control of extracellular adenosine levels. Infectious diseases can influence the synthesis of new cells or cause cell destruction, as occurs in canine rangeliosis, which results in anemia, thrombocytopenia, leukocytosis, and/or leukopenia. Thus, this study aimed to evaluate E-ADA activity in sera, erythrocytes, lymphocytes, and adenosine levels in sera samples of dogs infected by Rangelia vitalii. Twelve animals were divided into 2 groups: noninfected (n = 5) and infected by R. vitalii (n = 7). Animals were infected with 2 ml of blood containing the parasite, and parasitemia was estimated daily for 20 days by microscopic examination of peripheral blood smears. Blood collection was performed on days 0, 10, and 20 post-infection (PI) in order to evaluate the evolution of the disease. The blood collected was used to assess the activity of E-ADA. We observed an increase of E-ADA activity in sera (day 20 PI) and erythrocytes (days 10 and 20 PI) in the infected group (P < 0.05). E-ADA activity in lymphocytes was decreased on day 10, when the parasitemia was high, and increased after 20 days, when the number of circulating parasites was low. HPLC measured adenosine levels in the serum and found a reduction on days 10 and 20 PI. In conclusion, our results showed that E-ADA activity was altered in sera, lymphocytes, and erythrocytes of dogs experimentally infected by R. vitalii as well as the serum concentration of adenosine. These alterations may contribute to the pathogenesis of anemia and immune response in infected dogs.

Oxidative mechanisms of fish hepatocyte toxicity by the harmful dinoflagellate Cochlodinium polykrikoides.

Harmful Algal Blooms caused by the marine ichthyotoxic dinoflagellate Cochlodinium polykrikoides are responsible for mass mortalities of wild and farmed fish worldwide. In this research, we investigated the cytotoxic mechanisms of aqueous extract of C. polykrikoides on isolated Rainbow trout (Oncorhynchus mykiss) liver hepatocytes. Algal extract exposure with isolated trout hepatocytes caused hepatocyte membrane lysis, reactive oxygen species (ROS) formation, glutathione depletion, lysosomal membrane rupture, collapse of mitochondrial membrane potential, ATP depletion and increase in ADP/ATP ratio, cytochrome C release into the hepatocyte cytosol, and activation of caspases cascade. Anti-oxidants, free radical scavengers, mitochondrial permeability transition (MPT) pore sealing agents, microsomal oxidases inhibitors, ATP generators and lysosomotropic agents protected fish hepatocytes against C. polykrikoides. Fish hepatocyte toxicity was also associated with mitochondrial and lysosomal membrane injury. These events caused cytochrome C release from the mitochondrial intra-membrane space into cytosol. The cytochrome C release could trigger activation of caspase-3 and apoptosis.