It's a small world for parasites: evidence supporting the North American invasion of European Echinococcus multilocularis.

Echinococcus multilocularis (Em), the causative agent of human alveolar echinococcosis (AE), is present in the Holarctic region, and several genetic variants deem to have differential infectivity and pathogenicity. An unprecedented outbreak of human AE cases in Western Canada infected with a European-like strain circulating in wild hosts warranted assessment of whether this strain was derived from a recent invasion or was endemic but undetected. Using nuclear and mitochondrial markers, we investigated the genetic diversity of Em in wild coyotes and red foxes from Western Canada, compared the genetic variants identified to global isolates and assessed their spatial distribution to infer possible invasion dynamics. Genetic variants from Western Canada were closely related to the original European clade, with lesser genetic diversity than that expected for a long-established strain and spatial genetic discontinuities within the study area, supporting the hypothesis of a relatively recent invasion with various founder events.

A review on invasions by parasites with complex life cycles: the European strain of Echinococcus multilocularis in North America as a model.

In a fast-changing and globalized world, parasites are moved across continents at an increasing pace. Co-invasion of parasites and their hosts is leading to the emergence of infectious diseases at a global scale, underlining the need for integration of biological invasions and disease ecology research. In this review, the ecological and evolutionary factors influencing the invasion process of parasites with complex life cycles were analysed, using the invasion of the European strain of Echinococcus multilocularis in North America as a model. The aim was to propose an ecological framework for investigating the invasion of parasites that are trophically transmitted through predator­prey interactions, showing how despite the complexity of the cycles and the interactions among multiple hosts, such parasites can overcome multiple barriers and become invasive. Identifying the key ecological processes affecting the success of parasite invasions is an important step for risk assessment and development of management strategies, particularly for parasites with the potential to infect people (i.e. zoonotic).

Evaluation of an automated magnetic bead-based DNA extraction and real-time PCR in fecal samples as a pre-screening test for detection of Echinococcus multilocularis and Echinococcus canadensis in coyotes.

Efficient and sensitive diagnostic tools are essential for the study of the eco-epidemiology of Echinococcus species. We evaluated an automated magnetic bead-based DNA extraction commercial kit followed by qPCR (MB-qPCR), for the detection of Echinococcus multilocularis and Echinococcus canadensis in coyote (Canis latrans) fecal samples. The diagnostic sensitivity was determined by validating the method against the scraping, filtration, and counting technique (SFCT) for samples collected in Canada. From the 60 samples tested, 27 out of 31 SFCT positives samples for Echinococcus cestodes were positive in the MB-qPCR for E. multilocularis, with a sensitivity of 87.1% (95% CI 70.2 to 96.4%). Two samples were also positive for E. canadensis in the MB-qPCR and confirmed by morphological identification of adult worms. The agreement of the MB-qPCR and the SFCT was statistically significant with a kappa value of 0.67 (95% CI 0.48-0.85; p value < 0.001). The magnetic bead-based DNA extraction followed by qPCR proved to have a sensitivity comparable to the SFCT to detect E. multilocularis. Although the diagnostic sensitivity for E. canadensis was not estimated, MB-qPCR identified E. canadensis cases previously overlooked when using SFCT. We propose a combination of molecular and morphological identification using the MB-qPCR and the SFCT to detect both parasites, allowing for a more efficient large-scale surveillance, and detecting co-infections of Echinococcus species that can be difficult to identify when only based on morphology.

Deep amplicon sequencing highlights low intra-host genetic variability of Echinococcus multilocularis and high prevalence of the European-type haplotypes in coyotes and red foxes in Alberta, Canada.

Echinococcus multilocularis (Em) is a zoonotic parasite considered a global emergent pathogen. Recent findings indicate that the parasite is expanding its range in North America and that European-type haplotypes are circulating in western Canada. However, genetic analyses are usually conducted only on a few parasites out of thousands of individuals within each definitive host, likely underestimating the prevalence of less common haplotypes. Moreover, mixed infections with several mtDNA haplotypes in the same host have been reported, but their relative abundance within the host was never estimated. We aimed to 1) estimate the frequency of co-infections of different Em haplotypes in coyotes (Canis latrans) and red foxes (Vulpes vulpes) from western Canada and their relative abundance within the definitive hosts, 2) detect less prevalent haplotypes by sampling a larger proportion of the parasite subpopulation per host, and 3) investigate differences in the distribution of Em haplotypes in these main definitive hosts; foxes and coyotes. We extracted DNA from ~10% of the worm subpopulation per host (20 foxes and 47 coyotes) and used deep amplicon sequencing (NGS technology) on four loci, targeting the most polymorphic regions from the mitochondrial genes cox1 (814 bp), nad1 (344 bp), and cob (387 bp). We detected the presence of mixed infections with multiple Em haplotypes and with different Echinococcus species including Em and E. granulosus s.l. genotypes G8/G10, low intraspecific diversity of Em, and a higher abundance of the European-type haplotypes in both hosts. Our results suggest a population expansion of the European over the North American strain in Alberta and a limited distribution of some European-type haplotypes. Our findings indicate that deep amplicon sequencing represents a valuable tool to characterize Em in multiple hosts, to assess the current distribution and possible origins of the European strain in North America. The potential use of next-generation sequencing technologies is particularly important to understand the patterns of geographic expansion of this parasite.

Detecting co-infections of Echinococcus multilocularis and Echinococcus canadensis in coyotes and red foxes in Alberta, Canada using real-time PCR.

The continued monitoring of Echinococcus species in intermediate and definitive hosts is essential to understand the eco-epidemiology of these parasites, as well to assess their potential impact on public health. In Canada, co-infections of Echinococcus canadensis and Echinococcus multilocularis based on genetic characterization have been recently reported in wolves, but not yet in other possible hosts such as coyotes and foxes. In this study, we aimed to develop a quantitative real-time PCR assay to detect E. multilocularis and E. canadensis and estimate the occurrence of co-infections while inferring about the relative abundance of the two parasites within hosts. We tested DNA extracted from aliquots of Echinococcus spp. specimens collected from intestinal tracts of 24 coyote and 16 fox carcasses from Alberta, Canada. We found evidence of co-infections of E. multilocularis and E. canadensis in 11 out of 40 (27%) samples, with 8 out of 24 (33%) in coyote samples and 3 out of 16 (19%) in red fox samples. DNA concentrations were estimated in three samples with Cq values within the range of the standard curve for both parasites; two of them presented higher DNA concentrations of E. multilocularis than E. canadensis. The use of qPCR aided detection of co-infections when morphological discrimination was difficult and quantification of DNA for samples within the standard curve. This is the first molecularly confirmed record of E. canadensis in coyotes and the first evidence of co-infections of E. multilocularis and E. canadensis in coyotes and red foxes.

Corrigendum to 'Detecting co-infections of Echinococcus multilocularis and Echinococcus canadensis in coyotes and red foxes in Alberta, Canada using real-time PCR' [IJP: Parasites and Wildlife 7 (2018) 111-115].

[This corrects the article DOI: 10.1016/j.ijppaw.2018.03.001.].

Evaluation of the toxicity of Uncaria tomentosa by bioassays in vitro.

Aqueous extracts of Uncaria tomentosa (Willdenow ex Roemer and Schultes) DC. (Rubiaceae) ('Uña de gato'), were analyzed for the presence of toxic compounds in Chinese hamster ovary cells (CHO) and bacterial cells (Photobacterium phosphoreum). Toxicity was evaluated by four systems: Neutral red assay (NR), total protein content (KB), tetrazolium assay (MTT) and Microtox test. The extracts of U. tomentosa did not show toxicity in vitro at the concentrations tested. Testing in vitro could be a valuable tool for evaluating toxicity of medicinal plants.

In vitro cytotoxicity of guanylhydrazones (MGBG and PGBG) on cultured Chinese hamster ovary cells.

Guanylhydrazones, methylglyoxal bis(guanylhydrazone) (MGBG) and a new compound, phenylglyoxal bis(guanylhydrazone) (PGBG), interfering with polyamine biosynthesis have considerable potential for the use as antiparasitic and antitumor agents. The effect of these drugs on the cellular viability of Chinese hamster ovary cells was examined by in vitro neutral red assay. The time exposure and metabolic influence was studied. These compounds have a dose- and time-dependent cytotoxicity. The IC50 values were of 597.22 micrograms/ml and 1.77 micrograms/ml for MGBG after 3 and 24 h of incubation, respectively, and 380.50 micrograms/ml for PGBG after 24 h of incubation. The PGBG treatment during 3 h had no cytotoxic effect when the concentrations were lower than 3000 micrograms/ml. With the cytotoxicity assay used, we observed that the presence of S9 in cultured medium did not influence the cytotoxicity of these compounds.

[Malignant acanthosis nigricans. Presentation of a case and a study of its incidence]. / Acantosis nigricans maligna. Presentación de un caso e investigación de su frecuencia.

In the Regional Hospital "Victor Lazarte Echegaray" of Trujillo, Perú, it was made a study in order to determine the frequency of the acanthosis nigricans, associate to cancer (malignant acanthosis nigricans), during january 1978 to october 1983, and a case was founded in a patient of 62 years old, who had advanced adenocarcinoma gastric of one year and a half of evolution. The dermatosis was manifested with the neoplasia, which corresponds to a generalized form, with hyperkeratosis palmo plantar, with whole loss of the axillary hair and only part of the pubic region, the mucous membranes were not affected. The patient survived 3 months after the diagnostic were made.

Apoptosis induced by different doses of caffeine on Chinese hamster ovary cells.

Caffeine has been investigated for its potential mutagenic activity to bacteria, fungi and mammalian cells in culture, and at high concentrations it is also an inducer of apoptosis. Caffeine can exert acute cellular toxicity, including inhibition of cell growth and cell death, in Chinese hamster ovary cells. The aim of this study was to evaluate the cell survival and apoptotic or non-apoptotic effects of caffeine to different concentrations in Chinese hamster ovary cells (CHO-K1). These effects were evaluated by measuring cell viability, caspase 8 activity and fragmented DNA. This study suggests that the concentration of caffeine is of critical importance because high doses of caffeine induce apoptosis and low concentrations can act as an antioxidant. Previously, the cytotoxicity of caffeine was evaluated using a wide range of concentrations by the neutral red test. From this screening, adequate doses were selected to perform the caspase activity and fragmentation DNA studies. The potential antioxidant effect of caffeine was studied using tert-butyl-hydroperoxide as a free-radical generator. The repeatability was checked through three separate tests with the same concentration.

The antimitotic activities of some benzodiazepines.

Among 9 benzodiazepines, tested on the proliferation of synchronously dividing flagellate cells, only diazepam and medazepam can induce an accumulation of abnormal mitotic figures after 24 h of treatment. It seems that there is not a direct relation between the activity of benzodiazepines on the central nervous system and their ability to inhibit mitosis.

Cytotoxicity and mutagenicity of 6-aminonicotinamide in Chinese hamster ovary cells.

A strong teratogen-6-aminonicotinamide (6-AN)-was tested for its ability to induce cytotoxicity and mutagenicity in Chinese hamster ovary (CHO) cells. Tests were performed in the presence and absence of a metabolic activation system (S-9 mix). Cytotoxicity was evaluated in CHO cells by the total protein content. The two single-gene mutation systems in CHO cells have been investigated. Both involve evaluating the response of the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus and specific inhibitors of the cellular (Na2+K+)-ATPase using 6-thioguanine and ouabain as selective agents, respectively. From our results, 6-AN showed a higher cytotoxic effect at concentrations over 1 x 10(-1) mg/ml. Cytotoxicity was significantly different with and without S-9 mix. 6-AN was cytotoxic per se, however, when 6-AN was biotransformed, in the presence of S-9 mix no biological activity (cytotoxic) was detected. Non-significant mutagenic activity was detected with 6-AN in the presence and in the absence of the metabolic activation system.

Caspase-dependent proteolysis of integral and peripheral proteins of nuclear membranes and nuclear pore complex proteins during apoptosis.

We have studied the fate of the nuclear envelope (NE) in different human cells committed to apoptosis by different chemical agents. Using a battery of antibodies against marker proteins of the three domains of the nuclear envelope, namely lamin B (LB) for the lamina, transmembrane proteins LBR and LAP2 for the inner nuclear membrane, and nucleoporins p62, Nup153 and gp210 for the nuclear pore complexes (NPCs), we observed a selective and conserved cleavage of LB, LAP2 and Nup153. In lymphoid cells, the rate of cleavage of these markers was independent of the apoptosis inducing agent, actinomycin D or etoposide, and more rapid than in attached epithelial cells. While lamin B is cleaved by caspase 6, the protease responsible for the cleavage of LAP2 and Nup153 was probably caspase 3, since (1) cleavage of both proteins was specifically prevented by in vivo addition of caspase 3 inhibitor Ac-DEVD-CHO and (2) consensus sites for these caspases are present in both proteins. As LB, LAP2 and Nup153 are exposed at the inner face of the nuclear envelope and all interact with chromatin, we suggest that their cleavage allows both the detachment of NE from chromatin and the clustering of NPCs in the plane of the membrane, two conserved morphological features of apoptosis observed in this study.

[Structural modifications of secretory epithelial cells of the salivary glands induced by a carbohydrate-rich cariogenic diet in rats].

Rats receiving a carbohydrate rich cariogenic diet (Stephan 580) showed structural modifications of their salivary glands when compared to a control group. These observations suggested that a disturbance of the after birth organogenesis sequences occurred during the experimental period for the submandibular glands. The elaboration of elaboration of secretion granules was strongly decreased in the parotid acinar cells. Such an indirect action of the hyperglucidic diet could contribute to the carious process development by modifying the oral environment.

Effects of diazepam on mitosis and basal body duplication of synchronously dividing flagellate cells.

We report here that diazepam (Valium, Roche) has an inhibitory effect on proliferation of synchronously dividing cultures of Dunaliella, whenever the drug is added during the cell cycle. The drug induced an accumulation of abnormal mitotic figures after 17 to 24 hr of treatment with the chromosomes distributed around the nuclei and a monopolar spindle, although the pole-to-chromosome microtubules were well-formed. Diazepam seemed to affect mitosis by inhibiting the separation of the basal bodies, whose duplication was perturbed in a dose-related manner. After 48 hr of treatment, the nucleus returned to interphase without having undergone mitosis, but cytokinesis might have taken place. The mechanism of this action of diazepam on mitosis remains to be elucidated. However, it does not appear to act in the same way here as it does on the central nervous system.

Okadaic acid induces spindle lengthening and disrupts the interaction of microtubules with the kinetochores in metaphase II-arrested mouse oocytes.

The cell cycle is regulated by phosphorylation events via a cascade of protein kinases and phosphatases, but many of their substrates remain unknown. To study whether proteins of the metaphase II-arrested mouse oocyte meiotic spindle are substrates for phosphorylation events, we used okadaic acid (OA), a potent phosphatase inhibitor, upon fully mature spindles. Incubation of oocytes for 3 hr with 1 microM OA led to a dramatic lengthening of the spindle and a disorganization of the metaphase plate. Electron microscope studies revealed that this was due to a disruption of the interactions between the microtubules and the kinetochores. Biochemical analysis including MPM-2 immunoblotting and [32P]phosphate labeling of whole oocytes revealed several changes in the phosphorylation pattern following the OA treatment. Moreover, meiotic spindle purification or microtubule-associated proteins (MAPs) isolation showed that some of these phosphorylations occur on proteins associated with the microtubules or with structures closely related to the spindle. These results suggest that the changes occurring in the microtubule network during the cell cycle are partly due to the phosphorylation of some proteins associated with the microtubules.

Gp330 is specifically expressed in outer cells during epithelial differentiation in the preimplantation mouse embryo.

During preimplantation development of the mouse embryo, a layer of outer cells differentiates into a perfect epithelium, the trophectoderm. The divergence between the trophectoderm and the inner cell mass takes place from the 8-cell stage to the 64-cell stage and precedes their commitment at the blastocyst stage. In this work, we have investigated the expression of gp330, a 330 x 10(3) M(r) glycoprotein found in clathrin-coated areas of the plasma membrane of some epithelial cells characterized by a high level of endocytic activity. Our results show that gp330 is first synthesized in 16-cell stage embryos and that its appearance is restricted to outer cells until the blastocyst stage. Furthermore, its expression is repressed in inner cells at a post-transcriptional level, probably through the development of extensive cell-cell contacts.

A major posttranslational modification of ezrin takes place during epithelial differentiation in the early mouse embryo.

The preimplantation development of the mouse embryo leads to the formation of two populations of cells: the trophectoderm, which is a perfect epithelium, and the inner cell mass. The divergence between these two lineages is the result of asymmetric divisions, which can occur after blastomere polarization at compaction. The apical pole of microvilli is the only asymmetric feature maintained during mitosis and polarity is reestablished only in daughter cells that inherit all or a sufficient part of this pole. To analyze the role of ezrin in the formation and stabilization of the pole of microvilli, we isolated and cultured inner cell masses (ICM). These undifferentiated cells can differentiate very quickly into epithelial cells. After isolation of the ICMs, ezrin relocalizes at the cell cortex before the formation of microvilli. This redistribution occurs in the absence of protein synthesis. The formation of microvilli at the apical surface of the outer cells of ICM correlates with a major posttranslational modification of ezrin. We show here that this posttranslational modification is not controlled by a serine/threonine kinase but an O-glycosylation may partially contribute to it. These data suggest that ezrin has at least two roles during development. First, ezrin may be involved in the formation of microvilli because it localizes at the cell cortex before microvilli appear in ICMs. Second, ezrin may stabilize the pole of microvilli because it is modified posttranslationally when microvilli form.

Sister chromatid exchange induced by several types of coffees in Chinese hamster ovary cells.

Different brands of commercial caffeinated and decaffeinated coffees (roasted, high roast, blend ground, and instant coffees) were studied. These coffees were tested for their ability to induce sister chromatid exchanges (SCE) in CHO-K1 cells. Tests were performed in the presence and in the absence of a metabolic activation system (S-9 mix). Results were compared to the roasting procedure because genotoxic products could be formed from these processes. Our results indicate that caffeinated instant coffees showed higher genotoxic activity than decaffeinated coffees. Non-significant genotoxic activity was detected with the green coffee (unroasted). The highest increase of the frequency of SCE occurred when the caffeinated instant coffee was tested in the absence of metabolic activation system. The repeatability of the test was checked through three assays with the same sample.