Toxoplasma gondii in Ireland: seroprevalence and novel molecular detection method in sheep, pigs, deer and chickens.

Toxoplasma gondii is among the most studied parasites worldwide but there is not much information about it published in Ireland. The objectives of this study were to determine the seroprevalence of T. gondii in sheep, pigs, deer and chickens and the molecular detection of T. gondii DNA in muscle tissue. Serum samples were collected from these species at the time of slaughter at Irish abattoirs during 2007 and tested for anti-T. gondii antibodies using a commercial semi-quantitative latex agglutination test. Antibodies (titre ≥1 : 64) were found in 36% (105/292) sheep, 4.7% (15/317) pigs and 6.6% (23/348) deer. In chickens, 18% (65/364) had antibody titres, ranging between 1 : 5 and 1 : 1024. Significant (P ≤ 0.05) age-related differences in seroprevalence were found in adult sheep (58.1%) and pigs (23.1%). Significant gender differences in seroprevalence was also found in sheep with more females (43%) than males (22.4%) being positive. However, when adjusted for age through logistic regression gender was no longer significant. Seroprevalence was also evaluated on farm locations grouped to NUTS level 3, but the prevalence was too low to draw any statistical conclusions. Using a nested PCR, the presence of T. gondii DNA was detected in diaphragm samples from 3.6% (3/83) sheep, 13.0% (3/23) pig and 4.2% (3/71) deer. Meat digestion liquids from a Trichinella spp. survey in pigs were also used for the first time to detect T. gondii. Toxoplasma gondii DNA was detected in 50% (10/20) of pooled samples. This is the first in depth study of T. gondii seroprevalence in animals in Ireland and a novel method, using digestion liquid from pooled diaphragm samples, for PCR detection in pigs is described.

Molecular diagnosis of Toxoplasma gondii in pregnant women.

BACKGROUND: Novel approaches in the diagnostics of T. gondii have enabled a detection of the parasite in the amniotic fluid or blood of pregnant women. OBJECTIVE: The high titres of IgM and IgG antibodies against T. gondii are not always indicative of the presence of this parasite in pregnant women, therefore the molecular assays can be used to diagnose and genetically characterise T. gondii in amniotic fluids and blood samples. METHODS: The study analysed 15 samples of amniotic fluid and 1 sample of the blood from pregnant women suspected for toxoplasmosis. The serological ELISA test was used for the immunological study and molecular analyses, PCR at SAG2 locus followed by RFLP analysis were used for Toxoplasma gondii genotyping. CONCLUSION: Using PCR assay with TGR1E gene we have confirmed the presence of T. gondii in the blood of a pregnant woman. The parasite was typed as genotype I, belonging to virulent strains (Tab. 1, Fig. 2, Ref. 30).

Biomonitoring of polychlorinated biphenyls (PCBs) in heavily polluted aquatic environment in different fish species.

The distribution and concentrations of polychlorinated biphenyls (PCBs) were determined in fish species (European perch Perca fluviatilis, northern pike Esox lucius, pike perch Sander lucioperca, wels catfish Silirus glanus, common carp Cyprinus carpio, European eel Anguilla anguilla, freshwater bream Abramis brama, goldfish Carassius auratus, and roach Rutilus rutilus) in a heavily polluted water reservoir Zemplínska sírava (Slovakia). The study performed at two different time points 5 years apart (2004 and 2009) revealed serious PCB contamination of fish muscle tissue and significant interspecies as well as tissue-specific differences in PCB uptake by fish. Total PCBs broadly correlated with the trophic position of individual fish species within a food chain (P < 0.01). The concentrations were particularly high in predatory fish species, perch, pike, and pike perch (108.0, 90.1, and 113.0 mg kg(-1) lipid wt, respectively), but comparable PCB values were also found in non-predatory detrivorous freshwater bream (128.0 mg kg(-1) lipid wt). The lowest PCB values were surprisingly assessed in European eel (17.1 mg kg(-1) lipid wt). Tissue analysis showed the highest storage capacity of the liver (hepatopancreas in cyprinids) with maximum concentrations recorded found in northern pike (214.0 mg kg(-1) lipid wt) and freshwater bream (163.0 mg kg(-1) lipid wt). Negative correlations, mostly not significant, between the total PCB concentrations and fish weight were observed (P > 0.05). The study has shown that the kind of fish, its feeding habit, and specific conditions of the habitat are mutually interrelated factors that are responsible for significant variations in fish body burdens. A tendency to PCB biomagnification was also proved in some fish species of this water reservoir.

Field evaluation of an intravital diagnostic test of Echinococcus multilocularis infection in red foxes.

Echinococcus multilocularis parasitizes the small intestine of red foxes (Vulpes vulpes) and other carnivores, and has a wide distribution throughout the northern hemisphere. This cestode is the causative agent of human alveolar echinococcosis, a life-threatening helminth zoonosis. In 2000-2002, 2130 red foxes were examined for its presence in Slovakia, with a total prevalence of 30.7%. The data on occurrence were obtained by the combination of necropsy of small intestines from red foxes and coproantigen detection in faecal samples. The correlation between the number of detected specimens and the value of optical density of copro-ELISA test was found. When worm burdens were low (1-25 specimens) the sensitivity of the method was 31.3+/-8.64%, when worm burdens were >50 specimens, 81.8+/-0.66%, and with high worm burdens (>1000 specimens) the sensitivity reached 100+/-0.34%. E. multilocularis presence was detected using the nested PCR method from the eggs in the faecal samples with a 100% specificity. In epidemiological surveys of this zoonosis, it is of crucial importance to detect animals with a high level of infection, which are responsible for the bulk of environmental contamination. The advantage of copro-ELISA test lies in allowing the intravital diagnostics to be employed within the epidemiological survey of E. multilocularis occurrence in the protected and urban areas.

Morphological and genetic characterization of Echinococcus granulosus in the Slovak Republic.

The present study was undertaken to characterize more thoroughly aetiological agent of cystic echinococcosis that still represents a health problem in the Slovak Republic. Sequencing of the mitochondrial ND1 gene, Random Amplified Polymorphic DNA (RAPD) analysis and isoenzyme analyses were used to characterize genetically protoscolices of 37 isolates from pigs and cattle and two isolates from humans collected in different regions of the Slovak Republic. Solely the G7 genotype (pig strain) was identified by ND1 sequences in all 14 pigs isolate examined by this method. This genotype was also found in two human patients and in two cattle. The exclusive finding of the G7 variant has implications for the implementation of control strategies given its unique developmental and biological properties. Despite striking morphological variability, a low degree of the intra-strain variability was found in the Slovak Echinococcus granulosus isolates with the genetic methods used. This was particularly associated with the shape and size of hooks, number of testes and shape of ovaries and uterus. GPI and MDH enzymes were found suitable for the discrimination of G7 and G1 (=cosmopolitan sheep strain) variant.

Molecular evidence for the presence of a G7 genotype of Echinococcus granulosus in Slovakia.

Variability in Echinococcus granulosus is very important epidemiologically since strain characteristics may influence local patterns of transmission of hydatid disease. To classify the genotype presented in pig protoscoleces of the Slovak territory, a DNA-based approach has been used. Nucleotide sequences for a 471 bp region of the mitochondrial NADH dehydrogenase 1 (ND1) gene revealed a substantial affinity of isolates examined to the G7 genotype. Only a 0.9-3.4% sequence variation was recorded for E. granulosus samples compared with the reference G7 variant. To distinguish between G7 and G9 genotypes not differing in ND1 sequences, isolates were additionally examined by PCR-RFLP analysis of the nuclear ITS1 region. The resulting two-banded pattern is characteristic for the G7 strain. The data presented thus provides the first explicit evidence of the G7 genotype in the Slovak region.

Development and morphological variability of Echinococcus granulosus.

Characteristics are presented of Echinococcus granulosus strobila from dogs experimentally infected with protoscolices from a single cyst isolated from the liver of a domestic pig in a central Slovakian district. Adult cestodes are characterized mainly by 28-32 hooks highly variable in shape and size; 26-51 testes situated mostly throughout the proglottis (1 or 3 rows behind the vitelline gland); a lobate ovary; the shape of the uterus, which in 10% of proglottides is more or less spherical, in 62% is saccular with lateral sacculations of variable size, in 27% is saccular without lateral sacculations and in 1% is more or less tubular or strongly ramified; and an intermediately rapid rate of development lasting until between 40 and 44 days p.i. The present report analyzes the high degree of variability of the clone studied, referring to the knowledge on the characteristics of the pig, sheep, and cattle strains of E. granulosus and E. multilocularis species, with which some taxonomic characters of the clone representatives overlap.

Some enzymes of carbohydrate metabolism in Mesocestoides corti and Heterakis spumosa.

The activities of selected enzymes of carbohydrate metabolism were measured in tetrathyridia of Mesocestoides corti and in adult females and males of Heterakis spumosa. When the species were compared, only lactate dehydrogenase and phosphoenolpyruvate carboxykinase activities were considerably higher in M. corti. Activities of other enzymes were higher in H. spumosa, with malate dehydrogenase activity being considerably so. In H. spumosa, enzyme activity was higher, and succinate dehydrogenase markedly so in males, when compared with females. Tetrathyridia aged 170 and 210 days show relatively stable malate and lactate dehydrogenase activities, and mice of ICR and BALB/c strains are suitable for the maintenance of tetrathyridia.

Chitin synthesis in zygotes of Ascaris suum.

In Ascaris suum chitin is formed in the zygote immediately after oocyte fertilization, and its synthesis is completed in the eggs from the distal half of the uterus. Incorporation of radiocarbon [14C] glucose into chitin of the eggshell was 40-fold higher than incorporation of [14C] glucosamine. The same rank order also holds for the incorporation of label from these isotopes into the glycogen of the ovaries. A large part of the radiolabel was incorporated first into oocyte glycogen and only after fertilization was it incorporated into eggshell chitin. Actinomycin D inhibited chitin synthesis in the eggs from the distal half of the uterus and it significantly reduced incorporation of radiocarbon from glucose into chitin.

Properties and localization of chitin synthase in Ascaris suum eggs.

Investigations were carried out into the properties and localization of chitin synthase (EC 2.4.1.16) in connection with the formation of Ascaris suum egg-shells. The chitin synthase of eggs exhibited properties very similar to those found in other chitin-synthesizing organisms. It was stimulated by the presence of ATP, GlcNAc and Mg2+ with maximal activity at pH 7.0. In the phase of active chitin synthesis, during formation of the primary shell layers, chitin synthase was bound to the structures of the eggs. On completion of chitin synthesis in the eggs, the amount of the enzyme increased in the soluble fraction. Two forms of chitin synthase have been found in the eggs, namely an inactive (zymogenic) and an active form. Transformation of these two enzyme forms represents a key mechanism in the regulation of chitin synthesis in A. suum eggs.

Some precursors of glycogen in Ascaris suum females.

Intensity of incorporation of the labelled carbon from glucose, glucosamine and leucine into the glycogen of somatic cells and reproductive organs of Ascaris suum females has been studied. Glucose was most intensively incorporated into the growing oocytes and into the uterine epithelial cells, whereas its incorporation into muscle glycogen was found to result in lowest rates. Radiocarbons from glucosamine and leucine were incorporated into tissue glycogen of female worms much less than glucose. Incorporation of isotopes into glycogen of the eggs depended on the formation of their primary shells. The ways of incorporation of isotopes into glycogen of the eggs after formation of all the shell layers are discussed.