Optimized DNA-based identification of Toxocara spp. eggs in soil and sand samples.

BACKGROUND: Toxocara canis and Toxocara cati are globally distributed roundworms and causative agents of human toxocariasis, via ingestion of Toxocara eggs. Control of Toxocara infections is constrained by a lack of sensitive methods for screening of animal faeces and environmental samples potentially contaminated by Toxocara eggs. In this work, a pre-analytical method for efficient extraction of DNA from Toxocara eggs in environmental samples was set up using our previously validated T. canis- and T. cati-specific quantitative real-time polymerase chain reaction (qPCR). For this purpose, the influence of different methods for egg lysis, DNA extraction and purification for removal of PCR inhibitors were assessed on environmental samples. METHODS: To select the best egg disruption method, six protocols were compared on pure T. canis egg suspensions, including enzymatic lysis and thermal or mechanical disruption. Based on the selected best method, an analytical workflow was set up to compare two DNA extraction methods (FastDNA™ SPIN Kit for Soil versus DNeasy® PowerMax® Soil Kit) with an optional dilution and/or clean-up (Agencourt® AMPure®) step. This workflow was evaluated on 10-g soil and 10-g sand samples spiked with egg suspensions of T. canis (tenfold dilutions of 104 eggs in triplicate). The capacity of the different methods, used alone or in combination, to increase the ratio of positive tests was assessed. The resulting optimal workflow for processing spiked soil samples was then tested on environmental soil samples and compared with the conventional flotation-centrifugation and microscopic examination of Toxocara eggs. RESULTS: The most effective DNA extraction method for Toxocara eggs in soil samples consisted in the combination of mechanical lysis of eggs using beads, followed by DNA extraction with the DNeasy® PowerMax® Soil Kit, and completed with an additional DNA clean-up step with AMPure® beads and a sample DNA dilution (1:10). This workflow exhibited a limit of detection of 4 and 46 T. canis eggs in 10-g sand and 10-g soil samples, respectively. CONCLUSIONS: The pre-analytical flow process developed here combined with qPCR represents an improved, potentially automatable, and cost-effective method for the surveillance of Toxocara contamination in the environment.

Molecular data reveal cryptic speciation and host specificity in Toxascaris leonina (Nematoda: Ascarididae).

Toxascaris leonina (Ascarididae) is a cosmopolitan and polyxenical parasite whose host are canids and felids. To date, molecular phylogenetic studies included toxascarid representatives collected only from dogs or felids, therefore the intra-species differences between T. leonina collected from different host species has not been noticed. In this paper, we test the hypothesis of cryptic speciation in the T. leonina complex based on extended sequence data (ITS1, nad1, cox1) and individuals collected from dogs, felids and foxes. Phylogenetic analysis clustered T. leonina representatives into three well-supported clades depending on their host species, i.e. dogs and wolves, wild felids and foxes. Both genetic distances and the barcoding-gap analysis strongly support the species status of populations inhabiting different hosts. The results suggest additional genetic separation in felids. However, to determine the actual size of the Toxascaris complex, it would be necessary to analyse individuals collected from other canid and felid Toxascaris leonina host species.

Distribution and dynamics of soil contamination with Toxocara canis and Toxocara cati eggs in Poland and prevention measures proposed after 20 years of study.

The studies conducted in the years 1994-2013 allowed to determine the distribution of Toxocara spp. eggs in the soil of different types of urban and rural areas in Poland and to establish seasonal and multi-year dynamics of soil contamination with T. cati and T. canis eggs. Out of 3309 soil samples examined, 14.9% contained the eggs and the average density was 3.43 eggs/100g of soil. The level of soil contamination was the highest in cities, lower in villages, and lowest in small towns. In urban areas eggs of T. cati were found more frequently than of T. canis, while in rural areas the opposite was true. Both, in urban and rural areas, the sites most heavily contaminated with Toxocara spp. eggs were household surroundings (backyards). Recreation areas (parks, playgrounds, lake beaches) were significantly less contaminated. In older parts of Poznan, in backyards, soil contamination with the eggs was generally high each month, being lowest in July and highest in December and the prevalence of T. cati eggs was much higher than of T. canis. The prevention policy introduced in the city thus far - paying great attention to dog faeces but not to cats was unsatisfactory. After 18 years of such policy, the number of Toxocara spp. eggs dropped significantly but it was not a linear phenomenon - the intensity of soil contamination fluctuated. To be more effective, preventive measures should be preceded with discrimination of T. canis and T. cati eggs recovered in the soil and for this purpose the new molecular techniques should be applied. A survey conducted in the population in 2010 showed that people's awareness on the significance of pets in the transmission of dangerous zoonoses to humans was unsatisfactory. We recommend to expand the knowledge of the subject among the populace.

Differences in some developmental features between Toxoplasma gondii-seropositive and seronegative school children.

Knowledge about the influence of latent toxoplasmosis on development and general biological condition of children is scant and thus the aim of the present study was to investigate these aspects in some detail. We compare school children in rural area seropositive and seronegative to the apicomplexan parasite Toxoplasma gondii (Nicolle et Manceaux, 1908) in terms of their developmental age, body mass and body height, physical fitness and end-of-term grades. Additionally, we evaluated the risk factors of infection with T. gondii such as the presence of cats in the household and eating raw meat products. With IFAT and ELISA tests, the prevalence of anti-Toxoplasma antibodies was 41% (190 children examined) and the rate of infection was higher in girls (44%) than in boys (36%). No significant differences were observed in morphological features and physical fitness of examined children. In girls the level of developmental age measured with electrophoretical mobility of nuclei method was significantly higher and school performance significantly lower for those infected with Toxoplasma than for uninfected; this finding was particularly intriguing. Only boys who ate raw meat products were more likely to be seropositive. The study provides some new information on gender differences in reaction to Toxoplasma infection.

Potential risk of zoonotic infections in recreational areas visited by Sus scrofa and Vulpes vulpes. Case study--Wolin Island, Poland.

The relation between intestinal parasite prevalence in wild boars and red foxes and the sanitary condition of the soil in recreational estates were determined. The analysis was made based on 36 samples of boar faeces and 22 samples of fox faeces, collected in their habitat as well as 60 samples of soil from two recreational areas. Two methods were used for faecal samples--flotation and direct faecal smear; and flotation in NaNO3 for soil samples examination. Zoonotic nematode eggs were recovered from 25.5% of boar faecal samples; they were Ascaris suum (22.2%) and Trichuris suis (5.6%). Other parasites found were: Metastrongylus sp. (69.4%), Oesophagostomum sp., Strongyloides sp. (36.6%) and Physocephalus sp. (8.6%) as well as coccidia (69.4%). In fox faeces, zoonotic nematode eggs were recovered from 31.8% of samples, and they were Toxocara canis (27.2%) and Ancylostoma caninum (18.2%). Tapeworm eggs were found in 36.4% of samples including Taenia sp. (22.7%). The presence of Uncinaria stenocephala (45.5%), Capillaria sp. (36.4%), Trichuris vulpis (4.5%) and coccidia (40.1%) was also detected. It was shown that both, flotation and faecal smear, as mutually complementary should be used for higher rate of detection of parasites in faeces. No eggs of zoonotic helminths in soil from recreational areas were found despite these areas were accessible to wild animals and pets. This could be explained by characteristics of the soil (loose sand soil) as well as by behaviour of the parasite hosts in the examined areas.

[Helminths of hedgehogs, Erinaceus europaeus and E. roumanicus from Poznan region, Poland--coprological study].

Fecal samples from the hedgehogs, Erinaceus europaeus (15 samples) and E. roumanicus (44 samples) collected in the Poznan region of Poland were examined. Endoparasites were isolated using Sheather's flotation technique. The prevalence of parasitic infections in all hedgehogs examined was 74.6% (E. europaeus--73.3%; E. roumanicus--75.0%). Two species of parasites occurred in both species of hosts: Aonchotheca erinacei (60% and 80%) and Eucoleus aerophilus (6.7% and 15.9%). Brachylaima erinacei was found only in E. europaeus (33.3%) while Physaloptera sp. (13.6%) and Crenosoma striatum (4.6%) only in E. roumanicus. The intensity of infection varied from 1 to 371 eggs/1g of feces in E. europaeus and from 1 to 194 eggs/1g of feces in E. roumanicus. In both sexes the majority of infections were of low intensity, high intensity infections occurring only sporadically. No zoonotic species of endoparasites were recorded in these samples.

[A comparison of soil contamination with Toxocara canis and Toxocara cati eggs in rural and urban areas of Wielkopolska district in 2000-2005]. / Porównanie skazenia gleby jajami Toxocara canis i Toxocara cati w srodowisku wiejskim i miejskim w Wielkopolsce w latach 2000-2005.

The aim of the studies was to compare the degree of soil contamination with Toxocara canis and T. cati eggs in rural and urban areas depending on time of sampling and type of places examined. Material and methods. Over 2000-2005 a total of 538 soil samples from 3 villages and 368 from Poznan city (Poland) areas were examined for Toxocara spp. eggs. In spring 418 samples in rural areas and 184 samples in urban areas were collected and in autumn 120 and 184 respectively. The samples were examined using flotation technique in saturated sodium nitrate. The discrimination of T. canis and T. cati eggs was based on the size of eggs and transparency of shell layers. Results. The contamination of soil with Toxocara eggs was higher in the urban areas (19.8% positive samples) than in the rural ones (15.6% positive samples) and city or village-backyards were most heavily contaminated. Both, in the villages and in the city, the degree of soil contamination with eggs in spring and autumn was similar (17.6 and 14.8% positive samples respectively). T. cati eggs were much more prevalent in urban areas (97% of all eggs recovered) while T. canis in rural areas (84% of all recovered eggs). The share of T. canis and T. cati eggs in soil contamination did not depend on the time of sampling.

Recommended method for recovery of Toxocara and other geohelminth eggs from soil.

The flotation method elaborated for recovery of Toxocara and other geohelminth eggs from soil is described. Soil samples of about 500 ml volume are picked from 3-cm superficial layer of the ground. In the laboratory, 40 g of dry and sifted material is analysed according to following procedure: 1 h standing, 20 minutes shaking and 3 minutes centrifugation (1500 rpm) in 5% sodium hydroxide (NaOH), then centrifugation, like above, with H2O for washing the sample and next with the saturated sodium nitrate (NaNO3) for flotation the eggs. Specimen is prepared by placing a cover slip on the positive meniscus of the flotation liquid.