A step forward in the understanding of the presence and expansion of Echinococcus multilocularis in Eastern Europe using microsatellite EmsB genotyping in Poland

Alveolar echinococcosis is a severe zoonotic disease caused by the parasite Echinococcus multilocularis. In Europe, the lifecycle of this cestode is mainly sylvatic based on a prey-predator interaction between the red fox and small rodents as definitive and intermediate hosts, respectively. National surveillance of E. multilocularis in red foxes in Poland has reported a clear distinction between low endemic areas (from 2 to 5.7%) in the western half and high endemic areas (11.8 to 50.0%) in the eastern half of the country. A drastic increase of prevalence has been observed in the eastern half of Poland since the 2000's. Microsatellite EmsB genotyping was performed on 301 E. multilocularis worms from 87 foxes sampled throughout Poland, leading to identification of 29 EmsB profiles. The main profile, Pol19, was identified across the country and accounted for 44.9% of the worms collected. The conformity of 18 Polish profiles was established by comparison with previous profiles identified in Europe, but none corresponded to the most common European profiles. Poland was confirmed as a peripheral area of the main European focus, with more recent colonization by the parasite. The sharing of common profiles mainly by neighboring provinces was confirmed by a clustering analysis identifying four main groups. Expansion of the parasite in Poland in these four groups appears to be influenced by the situation in neighboring countries. Acquiring EmsB genotyping data from eastern European countries, for which very few data are reported, is necessary to understand the expansion of the parasite in the whole of Europe.     

Survey of Migratory Birds (Anatidae: Anas platyrhynchos) for Schistosome Parasites from Mazandaran Province,Northern Iran

Background

The aim of the present study was to survey birds’ schistosomes in migratory birds (Anatidae: Anas platyrhynchos) which are the source of the disease in Mazandaran Province, Northern Iran.

Methods

A number of mallards were bought from the markets of hunted birds. The respiratory tracts (nasal mucosa) and intestinal blood vessels were studied for adult worms. The nasal mucosa was separated and observed by a microscope. In order to separate the visceral schistosomes, after separating intestine, vessel mesenteric was studied under the lamp light and then in saline. The parasite sample was collected for subsequent observation.

Results

Fifteen (13.6%) cases out of 110 studied birds had nasal mucosa contaminated with Trichobilharzia sp. egg. Besides that, two birds had adult worms schistosome visceral i.e. Bilharziella sp.

Conclusion

The elements that cause cercarial dermatitis in aforementioned region are Trichobilharzia sp. and Bilharziella sp. parasites. Thus, it is necessary for the authorities of health, environmental and agricultural organization of the province to cooperate in order to control this disease.     

Tolerance to Zinc in Populations of the Earthworm Lumbricus rubellus from Uncontaminated and Metal-Contaminated Ecosystems

Zinc tolerance in Lumbricus rubellus populations from two metal-polluted (smelter and mine) sites was studied by comparing the effects of zinc with responses in a reference site strain. For the study, adult worms were collected directly from the field. Thus, no attempt was made to differentiate between tolerance resulting from population-level genetic adaptation or phenotypic plasticity in metal physiology. To compare relative sensitivity for zinc, worms from the three populations were exposed in laboratory tests. Effects on survival, weight change, cocoon production, and internal zinc levels were measured. Prior to exposure, it was anticipated that worms from the metal-contaminated sites would show substantially increased tolerance to zinc. This was not the case for all measured parameters. Thus, although differences in the shape of the dose-response relationships for survival and cocoon production were found, substantial variations in measured responses, effect concentrations, or zinc accumulation rates were not apparent. Overall, therefore, zinc tolerance is unlikely to be a major factor influencing the distribution of L. rubellus in contaminated regions. Received: 8 September 1998/Accepted: 21 March 1999     

Larva Migration and Eosinophilia in Mice Experimentally Infected With Gnathostoma spinigerum

Background

Gnathostoma spinigerum causes larva migran in human which is endemic in Southeast Asia. Information regarding larva migration is limited. In this study, we investigated the parasite migration by recovery of worms from the whole body of mouse after oral infection with advanced third stage larvae (AL3). The percentage of blood eosinophils was examined in parallel.

Methods

Mice were orally infected with AL3 and histological study of organs was investigated in order to study the migration of AL3, along with blood eosinophilia.

Results

At 1 hr post infection (PI), the larvae remained in the stomach, thereafter at 3, 5, 7, 10 and 24 hr PI; they were recovered from various organs including liver, mesentery, esophagus, diaphragm, lung, heart and dorsal fat. At day 15 PI, they were mostly found in muscles (76.47%). The average worm recovery (5 months) was 78.03%. The worms were found in the liver at every time point. Larva encystment was detected. There was a significant difference in blood eosinophils between the 8 larvae- (average 9.33% + 6.25%) and the 15 larvae-infected groups (average 22.66% + 11.03%). Surprisingly, the blood eosinophils (average 19.00% + 2.92%) were not higher in the higher infective dose- group (25 larvae).

Conclusion

Liver was involved by G. spinigerum throughout the study. We detected larva encystment which had never been reported in human gnathostomiasis. The highest percentage of eosinophil occurred during the invasive stage.     

Application of organic wastes on a benzo(a)pyrene polluted soil. Response of soil biochemical properties and role of Eisenia fetida

In this paper we studied the bioremediation effects of a soil artificially contaminated by benzo(a)pyrene with and without two organic wastes (organic municipal solid waste, MSW, and poultry manure, PM) and with and without worms (Eisenia fetida) over 90 days. For the organic treatments, soil samples were mixed with MSW at a rate of 10% or PM at a rate of 7.6%, in order to apply the same amount of organic matter to the soil. An unamended and non-polluted soil was used as control. Cellulase and glutathione-S-transferase activities in worms and the earthworms’ weight were measured at four different incubation times (3, 15, 60 and 90 days). Cocoon numbers, average weight per cocoon and number of juveniles per cocoon were measured 30 days after the benzo(a)pyrene exposure. Extractable benzo(a)pyrene in soils and E. fetida was determined during the incubation period. To observe the effects of bioremediation of the contaminated soil, ATP, urease and phosphatase activities were measured. At the end of the incubation period and when compared with the polluted soil without worms and organic matter, the extractable benzo(a)pyrene decreased by 41.2% for the unamended polluted soil and without worms, by 45.8% for the organic-PM polluted soil and without worms, 48.3% for the organic-MSW polluted soil and without worms, 55.4% for the organic-PM polluted soil and with worms, and 66.3% for the organic-MSW polluted soil and with worms. This meant that worm hydrocarbon absorption was lowest in the contaminated soil amended with MSW and with worms, causing an increase in catabolic activity of the soil. These results suggested that the co-application of organic wastes and E. fetida for the bioremediation of benzo(a)pyrene polluted soil is potentially advantageous.     

Establishment of a Modified in Vitro Cultivation of Protoscoleces to Adult Echinococcus granulosus; an Important Way for New Investigations on Hydatidosis

Background

Echinococcus granulosus, a zoonotic cestode parasite, causative agent of hydatid cyst is endemic in many parts of the world including the Middle East. Study on different aspects of this parasite is very important and valuable. However, working with adult worms which their habitat situated in the small intestine of canids, is dangerous and risky. Achieving such risky situation needs a controlled condition which is cultivation of the organisms in the laboratory. In this regard, cultivation of E. granulosus protoscoleces leading to adult worms was established in the laboratory for the first time in Iran.

Methods

Under aseptic conditions a number of protoscoleces were cultivated in diphasic S.10E.H medium using CO2 incubator to produce adult worms.

Results

Different forms of parasites including pre-segmentation stages (PS1 - PS4) and segmentation stages (S5-S8) and developing stages in segmented worms (S10-S11) were observed and evaluated in these medium. Finally adult worms contained four proglottids with a large and distinct genital pore were observed 50-55 days post cultivation. These parasites do not produce fertile eggs and conclusively do not have risk of hydatid disease transmission to the researchers.

Conclusion

The mentioned method for producing E. granulosus adult worms can open a new window for researches and facilitate working on different aspects of hydatidosis especially for diagnosis, protection and treatment studies.     

Sperm Morphology and Motility of <Emphasis Type="Italic">Bufo raddei</Emphasis> Under Long-Term Environmental Heavy Metal Stress

The present study aimed to investigate the effects of environmental heavy metals on sperm morphology and motility of Bufo raddei. We collected B. raddei from Baiyin (BY) and Liujiaxia (LJX); BY is an area heavily polluted by heavy metals, while LJX is a relatively unpolluted area. Significant accumulations of copper, zinc, lead and cadmium were observed in B. raddei from BY, together with relatively smaller sperm size and higher sperm abnormality rate. Unexpectedly, sperm velocity, percentages of sperm motility and viability of B. raddei from BY were significantly higher than those from LJX. Similarly, when sperm was suspended in heavy metal solutions, sperm velocity, percentages of sperm motility and viability significantly increased compared with those suspended in distilled water. These results indicate that environmental heavy metal stress may decrease sperm size, meanwhile enhance sperm motility through increasing the ratio of head length to total sperm length of B. raddei.     

Supplement Timing of Cranberry Extract Plays a Key Role in Promoting Caenorhabditis elegans Healthspan

Consumption of nutraceuticals is a major and potent dietary intervention for delaying aging. As the timing of administration is critical for the efficacy of bioactive compounds in medicine, the effectiveness of nutraceuticals may also be dramatically affected by the timing of supplementation. Cranberry exact (CBE), rich in polyphenols, is consumed as a nutraceutical, and possesses anti-aging properties. Here, we examined the influence of timing on the beneficial effects of CBE supplementation in C. elegans. The prolongevity effect of CBE in different aged worms, young adults, middle-age adults, and aged adults, was determined. Early-start intervention with CBE prolonged the remaining lifespan of worms of different ages more robustly than late-start intervention. The effectiveness of CBE on stress responses and physiological behaviors in different aged worms was also investigated. The early-start intervention prominently promoted motility and resistance to heat shocks and V. cholera infection, especially in aged worms. Together, these findings suggest that the timing of CBE supplementation critically influences its beneficial effects on C. elegans lifespan and healthspan. It is of interest to further investigate whether the similar results would occur in humans.     

Toxocara Nematodes in Stray Cats from Shiraz,Southern Iran: Intensity of Infection and Molecular Identification of the Isolates

Background

Toxocara is a common nematode of cats in different parts of Iran. Despite the close association of cats with human, no attempt has been done so far for molecular identification of this nematode in the country. Therefore, current study was performed on identification of some isolates of Toxocara from stray cats in Shiraz, Fars Province, Southern Iran, based on morphological and molecular approaches, and also determination of intensity of infection.

Methods

This cross-sectional study was carried out on 30 stray cats trapped from different geographical areas of Shiraz in 2011. Adult male and female worms were recovered from digestive tract after dissection of cats. Morphological features using existing keys and PCR-sequencing of ITS-rDNA region and pcox1 mitochondrial l gene were applied for the delineating the species of the parasites.

Results

Eight out of 30 cats (26.7%) were found infected with Toxocara nematodes. All the isolates were confirmed as Toxocara cati based on morphological features and the sequence of ribosomal and mitochondrial targets. Intensity of infection ranged from one to a maximum of 39 worms per cat, with a mean of 10.25±12.36, and higher abundance of female nematodes.

Conclusion

The most prevalent ascaridoid nematode of stray cats in the study area was T. cati and female nematodes were more abundant than that of males. This issue has important role in spreading of eggs in the environment and impact on human toxocariasis.     

Effectiveness of the whole-cell pertussis vaccine produced in Poland against different Bordetella parapertussis isolates in the mouse intranasal challenge model

The aim of the study was to evaluate the effectiveness of the whole-cell pertussis vaccine produced locally and routinely used in Poland in the elimination of Bordetella parapertussis strains from the lungs and trachea of a mouse model. We found that the average protective effect against B. parapertussis in the lungs of mice immunized with the whole-cell pertussis vaccine (DTwP) was significantly higher than in animals immunized with the acellular pertussis vaccine (DTaP). The effectiveness of B. parapertussis elimination rates from the lungs of DTwP-immunized mice, depending on the strain used as a challenge, was found to be 1.2-3.0 times or 3.1-7.0 times lower than against Bordetella. pertussis Tohama I or vaccine B. pertussis 606/67 isolates, respectively. Our results show that the locally produced DTwP vaccine is able to protect against B. parapertussis isolates; however, the level of protection and course of B. parapertussis infection in the lungs and trachea seems to be strain specific.     

Bioaccumulation of Some Heavy Metals in the Liver Flukes Fasciola hepatica and F. gigantica

Background

Heavy metals tend to bioaccumulate in living organisms, and their accumulation has been a major concern. As mammals are known to excrete heavy metals via their bile, it seems to be very promising to analyse metal burdens of parasites that infect the biliary tree such as liver flukes of the genus Fasciola. The present study was carried out to evaluate F. hepatica and F. gigantica as bioaccumulators of heavy metals, and to estimate their use as sensitive markers of environmental pollution with heavy metals.

Methods

A total of 36 slaughtered buffaloes (26 infected and 10 controls) collected from the slaughter-house of Tanta City, Egypt were used. Samples of muscle and liver tissues were taken from each buffalo. A total of 44 adult Fasciola flukes were collected from the 26 infected buffaloes. Quantification of some heavy metals (Cd, Cr, Cu, Pb and Zn) in samples was carried out using electrothermal atomic absorption spectrometry.

Results

Results revealed different concentrations of heavy metals in different host tissues. The adult flukes were classified into F. hepatica (n = 25) and F. gigantica (n = 19). The bio-concentration factor (BCF) of Cr was significantly higher in F. hepatica (P = 0.0465) while BCF of Zn was significantly higher in F. gigantica (P = 0.0189). A comparative study between the two species as regards the BCF was never done before.

Conclusion

The obtained results indicate the possibility of use of Fasciola flukes as markers of environmental pollution with some heavy metals.     

A molecular epidemiological investigation of Ascaris on Unguja, Zanzibar using isoenyzme analysis, DNA barcoding and microsatellite DNA profiling

Ascariasis is of public health importance on the islands of Zanzibar (Unguja and Pemba). To shed light on the molecular epidemiology of this parasite, 68 Ascaris worms, obtained from 14 individuals in four Ungujan villages, were examined by isoenzyme analysis (ISA), DNA barcoding and microsatellite DNA profiling. ISA revealed genetic variation, which was confirmed by DNA barcoding. Nineteen worms recovered from individuals in Uganda were included for comparison. Sixteen unique DNA barcodes were identified, 15 on Unguja and three in Uganda with two shared between. These two barcodes were found in all four Ungujan villages. Worms from Tumbatu-Jongowe, an isolated village on an islet off Unguja, seemed particularly diverse. Within our barcodes, three exact matches were found with Chinese Ascaris retrieved from pigs, which is perhaps surprising given the present rarity of these animals on Unguja. Microsatellite profiling and population genetic analysis revealed further genetic diversity within our samples although population sub-structuring within Unguja was minor in comparison to that between Unguja and Uganda. As African Ascaris has not been subjected to detailed molecular scrutiny, this new diversity represents an important piece in its evolutionary jigsaw and such population markers are informative in monitoring worm dynamics during ongoing control.     

Evidence for mitochondrial pseudogenes (numts) as a source of contamination in the phylogeny of human whipworms

Trichuris trichiura and T. suis are whipworms of humans and pigs, respectively, but it has recently been suggested that humans may be infected with multiple genotypes or species of Trichuris and cross-infection with Trichuris of pig origin has also been reported. In addition, the species status of Trichuris in non-human primates is unsettled and it is unknown how many whipworm species we share with other primates. Herein, we inferred the phylogeny of Trichuris collected from human, baboon and pig based on nuclear (18S and beta-tubulin) and mitochondrial (cox1) genes and evaluated the use of three PCR linked restriction fragment length polymorphism (PCR-RFLP) to identify worms. We found that all baboon worms clustered with human worms and that all these primate worms are different from T. suis. In general, there was an agreement between the phylogeny established based on the nuclear and mtDNA genes. However, we found evidence for non-targeted cox1 gene amplification for a subset of the human worms and suggest the presence of mitochondrial pseudogenes (numts) of pig cox1 gene in the human Trichuris genome. In conclusion, phylogenetic characterization of human whipworm based on the cox1 gene alone may be problematic without suitable preceded measures to avoid the numts amplification.     

Still and Moving Image Evidences for Mating of Echinococcus granulosus Reared in Culture Media

Background

Echinococcus granulosus cultivation is very important for improvement of different aspect of medical and veterinary researches. Despite many advances in this case, there is a missing link for in vitro life cycle of adult worms and it is fertilization. Regarding the researchers’ observations, self-fertilization can be done in worms living in dog intestine, but despite all sorts of experimental techniques, this phenomenon has never been observed in reared worms in culture media. Furthermore, cross fertilization has not been observed in vitro and even in parasites with dog intestinal origin; although it theoretically is possible. During a follow-up of cultivated adult worms, evidences of behaviors similar to self-mating (Type 2) and cross-mating were observed in our lab which will be presented here.

Methods

Protoscoleces were aseptically removed from sheep hydatid cysts, washed twice with PBS and then cultivated in S.10E.H culture medium. The stages of parasite growth were observed using an inverted microscope for two months and all stages and behaviors were microscopically photographed. Different movies have also been made from these behavioral features.

Results

After around 55 days post cultivation, some evidences of behaviors similar to self-mating (Type 2) and cross-mating were observed in some of the mature adult worms. However, fertile eggs in these parasites have never been observed.

Conclusion

Regarding the above observations, these parasites show tendency to unsuccessful self-mating/fertilization (type 2) which failure could be due to anatomical position and physiological maturation. Also lack of suitable conditions for self-fertilization causes the worms try to do unsuccessful cross- mating/fertilization in culture media     

Experimental Life Cycle of Hypoderaeum conoideum (Block, 1872) Diez, 1909(Trematoda: Echinostomatidae) Parasite from the North of Iran

Background:

Human Echinostomiasis is an intestinal disease caused by the members of family Echinostomatidae parasites. The aim of present research was to identify echinostomatidae cercariae emitted by Lymnaea palustris snails from Mazandaran province in the north of Iran based on the morphological and morphometrical characteristics of the different stages of experimental parasite life cycle.

Methods:

Echinostomatidae cercariae were collected from L. palustris (Gastropoda: Lymnaeidae) of the north of Iran. To collect metacercaria, 50 healthy snails were infected with cercariae experimentally (50 cercariae for each). To obtain the adult stage, 9 laboratory animals (3 ducks, 2 rats, 2 mice and 2 quails) were fed with 60 metacercaria for each. To identify parasite, the different stages of worm were examined using light microscope and then the figures were draw under camera Lucida microscope and measures were determined.

Results:

Averagely, 15metacercaria were obtained from each snail that had been previously exposed with cercariae. Ducks presented worm eggs in feces after 10–15 days post-infection. Intestinal worms were collected and identified as Hypoderaeum conoideum on the bases of figures and measures of cephalic collar, the number of collar spine, suckers diameter ratio, testes arrangement, etc.

Conclusion:

H. conoideum cercariae and adult worm are described. This is the first report of the different stages of the experimental life cycle of this parasite in Iran.     

Heavy Worm Burden of Moniliformis moniliformis in Urban Rats with Histopathological Description

Background

Due to scarcity of human reports, we took advantage of the heaviest infection of M. moniliformis in rats, to describe histopathological and microanatomical valuable useful keys while confronting human occurrences.

Methods

Samples were obtained from captured rats in Tehran, capital of Iran, during two decades. Tissue sections were performed through hematoxylin and eosin staining to describe histopathological changes in rat''s intestines.

Results

Totally, nine rats were found infected with M. moniliformis amongst 272 obtained rats. Heavy infection has been distinguished in 2 individuals with parasite burden of 141and 73 adult worms. Cross sections of worms within the lumen show mucosal thickness, infiltration of eosinophilic leukocyte and increase in goblet cells.

Conclusion

Beyond the uncommonness of human infection with M. moniliformis unitended infections should not be ignored. Abundance of rats and roaches as definite and intermediate hosts must be considered particularly in countries with poor hygiene.     

Toxoplasma Infection in Schizophrenia Patients: A Comparative Study with Control Group

Background

Schizophrenia is a serious, chronic, and often debilitating neuropsychiatric disorder. Its causes are still poorly understood. Besides genetic and non-genetic (environmental) factors are thought to be important as the cause of the structural and functional deficits that characterize schizophrenia. This study aimed to compare Toxoplasma gondii infection between schizophrenia patients and non-schizophrenia individuals as control group.

Methods

A case-control study was designed in Tehran, Iran during 2009-2010. Sixty-two patients with schizophrenia and 62 non-schizophrenia volunteers were selected. To ascertain a possible relationship between T. gondii infection and schizophrenia, anti-Toxoplasma IgG antibodies were detected by indirect-ELISA. Data were statistically analyzed by chi- square at a confidence level of 99%.

Results

The sero-positivity rate among patients with schizophrenia (67.7%) was significantly higher than control group (37.1) (P <0. 01).

Conclusion

A significant correlation between Toxoplasma infection and schizophrenia might be expected.     

Quantitative Ultrastructure of Metal-Sequestering Cells Reflects Intersite and Interspecies Differences in Earthworm Metal Burdens

Morphometric analysis of transmission electron micrographs was used to compare the effects of metals on the multifunctional, metal-sequestering, chloragocyte cells of two epigeic earthworm species, Dendrodrilus rubidus and Lumbricus rubellus, inhabiting three field soils: a clean circumneutral reference soil (Dinas Powys); an acidic moderately Pb- and Zn-contaminated soil (Cwmystwyth); and a calcareous Cd-, Pb-, and Zn-contaminated soil (Draethen). The main findings were: (1) D. rubidus accumulated significantly higher tissue Cd and Pb and lower Zn concentrations than L. rubellus, especially at Draethen; (2) the volume fraction of chloragosomes was significantly lower and the volume fraction of debris vesicles significantly higher in D. rubidus from Draethen compared with L. rubellus at all sites and with the other two D. rubidus populations; (3) estimated relative toxicity factors, derived from soil metal concentrations and published EC₅₀ data, suggested that the subcellular changes in chloragocytes, particularly in D. rubidus from Draethen, were caused mainly by Zn and Pb exposures; (4) scrutiny of the body burdens of each metal in both worm species across the three sites indicated that Cd was a major contributor to the structural changes observed in Draethen D. rubidus, and its impact was disproportionate to its soil and tissue concentrations in comparison with those of Pb and Zn. The apparent greater susceptibility of D. rubidus cells, compared with L. rubellus cells, to soil metal contaminants is discussed in light of differences in the quality and quantity of the metal body burdens accumulated by the two species. Further histopathalogic and morphometric studies on key organs and tissue of earthworms are required to provide biomarkers of exposure and to underpin linkage of biochemical-level changes and demography.