Cyanobacterial cell-wall components as emerging environmental toxicants - detection and holistic monitoring by cellular signaling biosensors.

Cyanobacterial blooms constitute a recognized danger to aquatic environment and public health not only due to presence of main group of cyanotoxins, such as microcystins, cylindrospermopsin or anatoxin-a, but also other emerging bioactivities. An innovative approach identifying such bioactivities is the application of cellular biosensors based on reporter genes which detect the impact of cyanobacterial cells and components on actual human cells in a physiological-like setting. In the present study biosensor cell lines detecting four different types of bioactivities (ARE - oxidative stress, NFKBRE - immunomodulatory pathogen-associated molecular patterns, AHRE - persistent organic pollutants, GRE - endocrine disruptors) were exposed to concentrated cyanobacterial cells from 21 environmental bloom samples and from eight cultures (Microcystis aeruginosa, Aphanizomenon flos-aquae, Planktothrix agardhii and Raphidiopsis raciborskii). The AHRE and GRE biosensors did not detect any relevant bioactivity. In turn, ARE biosensors were significantly activated by bloom samples from Jeziorsko (180-250%) and Sulejów (250-400%) reservoirs with the highest cyanobacterial biomass, while activation by cultures was weak/undetectable. The same biosensors were stimulated by microcystin-LR (250%) and anatoxin-a (150%). The NFKBRE biosensors were activated to varying extent (140-650%) by most bloom and culture samples, pointing to potential immunomodulatory toxic effects on humans. Lipopolysaccharide and lipoproteins were identified as responsible for NFKBRE activation (probably via pattern recognition receptors), while peptidoglycan had no bioactivity in this assay. Thus, the holistic approach to sample analysis with the application of cellular biosensors geared towards 4 separate pathways/bioactivities was validated for identification of novel bioactivities in organisms with recognized public health significance (e.g. this study is the first to describe cyanobacterial lipoproteins as potential environmental immunomodulators). Moreover, the ability of cellular biosensors to be activated by intact cyanobacterial cells from blooms provides proof of concept of their direct application for environmental monitoring, especially comparison of potential threats without need for chemical analysis and identification of toxicants.

Microarray analysis of rat immune responses to liver fluke infection following vaccination with Fasciola hepatica phosphoglycerate kinase.

Fasciolosis is a considerable veterinary problem, causing significant economic losses to livestock production and the food industry. Research in the area of Fasciola hepatica infection immunology is necessary to improve our knowledge about immunological mechanism evoked by the parasite and to develop new control strategies against liver fluke. In this present paper we analyzed the expression levels of cytokines in rats infected with F. hepatica following immunization with F. hepatica phosphoglycerate kinase - a novel vaccine antigen. Immune response analysis using microarray was undertaken six weeks after infection. Expression levels of INF-γ and IL-4, which are characteristic cytokines secreted during Th1-like and Th2-like immune responses, respectively, were unchanged in vaccinated animals as compared to control animals. This indicates the vaccine did not influence the major modulation of immune responses typically observed during Fasciola infections, however, other subtle but significant variations were observed that indicated altered inflammatory and possibly T helper cell responses. A significant rise in IL-12α chain expression levels was observed. Expression levels of TNF-α and some related molecules, such as ADAM17, FasL, CD40 and TRAF3 were also elevated. Expression levels of molecules involved in IL-1 signaling pathways were reduced, although a rise in IL-1α expression was noted.

Detection of Giardia intestinalis assemblages A, B and D in domestic cats from Warsaw, Poland.

Giardia intestinalis is a complex species divided into 7 assemblages (A - G). Two of them (A and B) are infective for both humans and animals. In cats four assemblages can occur: A, B, D, and F Assemblages A and B infect either cats, dogs and humans, assemblage D infects cats and dogs and assemblage F only cats. The purpose of this study was to determine the prevalence and genotypes of G. intestinalis in cats from Warsaw. From November 2006 to March 2007 a hundred sixty samples of stool were collected and examined by light microscopy. G. intestinalis cysts were detected in 3.75% of samples. DNA extracted from positive samples was used as template for PCR-RFLP using Giardia specific primers and the amplicons were sequenced. A comparison of the obtained DNA sequences with the Giardia sequences in the GeneBank database revealed assemblage A in 1.25% of the investigated cats, assemblage B in 1.25% and D in 1.25%.

Blocking Fasciola hepatica's energy metabolism - a pilot study of vaccine potential of a novel gene - phosphoglycerate kinase.

Fasciola hepatica infections cause huge economic losses to livestock production and are a serious problem in human and veterinary medicine. The main difficulty in the control of infections is progressing drug resistance. Moreover, pharmacological therapy is expensive and harmful for the environment. The best way of prophylaxis against infections seems to be vaccination. A new generation of vaccines could be the best possible way of controlling fasciolosis. This paper is focused on first vaccination trials based on a new vaccine candidate antigen, F. hepatica phosphoglycerate kinase (FhPGK) performed on a rat model. We obtained protection levels ranging from 0% to 69% depending on the way of delivery and form of vaccine.

Hematological changes during the course of canine babesiosis caused by large Babesia in domestic dogs in Warsaw (Poland).

In the presented study we evaluated the hematological changes in samples of blood obtained from 248 dogs naturally infected with large Babesia. The evaluation included red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), leucocyte counts, thrombocyte counts, mean platelet volume (MPV), morphology of erythrocytes and leucogram. The most common disorders in affected dogs were thrombocytopenia and anisocytosis. The count of erythrocytes below reference values was detected in 26.2% of dogs and 31.4% of affected animals presented hematocrit below the reference values. Hemoglobin concentration below the reference values was noted in 29% of dogs, an increase of MCHC above normal values was detected in 21% of examinated dogs and MCV below normal values was recognized in 2% of dogs. 60.5% of dogs presented anisocytosis, 25% poikilocytosis, 23.8% polychromasia, 19.7% hypochromia and 4.4% erythroblastosis. Thrombocytopenia was detected in 99.5% of dogs, but only 15.3% of examined animals showed increase of MPV, which suggests a response of the bone marrow. 36.3% of dogs had neutropenia, and 21.8% presented a left shift, 14.9% had the lymphocytosis and 7.2% lymphopenia.

[Prevalence of Giardia intestinalis in domestic dogs in Warsaw]. / Wystepowanie Giardia intestinalis u psów domowych w Warszawie.

INTRODUCTION: Giardia intestinalis is the most common intestinal protozoan parasite, which infects humans, dogs and other mammals throughout the world. So far eight genotypes of the parasite have been described of which four were found in dogs. Assemblages A-I and B infect either dogs or humans. Assemblages C and D occur only in dogs. The aim of the study was to determine the prevalence and genotypes of G. intestinalis in domestic dogs of Warsaw area. MATERIAL AND METHODS: From October 2005 to March 2006 fecal samples were collected from 350 dogs and examined using light microscopy and PCR techniques. RESULTS: 5.14% of dogs was found to be positive for G. intestinalis by microscopy and prevalence of 9.14% was found by PCR. The PCR amplicons were sequenced and the DNA sequences were compared with Giardia sequences in GeneBank database. The analysis revealed assemblage A-I in 1.71% of dogs, assemblage C in 1.14% and assemblage D in 6.28% of dogs in Warsaw. According to literature, the genotype A can infect humans however a role of dogs as a reservoir of human giardiosis in Poland is not known.

[Molecular and morphological comparison of hookworms from genus Uncinaria invading red fox (Vulpes vulpes) and dog (Canis familiaris)]. / Morfologiczne i molekularne porównanie nicieni z rodzaju Uncinaria pasozytujacych u lisa (Vulpes vulpes) i psa (Canis familiaris).

Two species of hookworms from genus Uncinaria have been found so far in Poland. Uncinaria stenocephala infects mainly dog, wolf and red fox, whereas Uncinaria criniformis is a parasite of mustelids (but it was also reported from red fox). 19 male and 29 female hookworms from red foxes have been compared with 10 male and 12 female worms from dogs. Hookworms from dogs were generally smaller than these from foxes, but no other morphological differences could be found. These hookworms were qualified to species Uncinaria stenocephala on the ground of morphology of male. Genomic DNA samples have been isolated from these hookworms and segments of rDNA including part of small subunit of ribosomal RNA gene; internal transcribed spacer 1 (ITS1); 5.8 S ribosomal RNA; internal transcribed spacer 2 (ITS2) and part of large subunit of ribosomal RNA have been amplified and sequenced. Sequences from Uncinaria obtained both from foxes and dogs have shown very high similarity to the sequence of Uncinaria stenocephala, so all examined hookworms have been classified as belonging to this species.