Identification of taeniid eggs in the faeces from carnivores based on multiplex PCR using targets in mitochondrial DNA

A multiplex polymerase chain reaction (PCR) was evaluated for the identification of morphologically indistinguishable eggs of the taeniid tapeworms from carnivores using primers targeting mitochondrial genes. The primers for Echinococcus multilocularis (amplicon size 395bp) were species-specific as assessed by in silico analysis and in the PCR using well-defined control samples. The design of primers that specifically amplify DNA from E. granulosus or Taenia spp. was not possible. The primers designed for E. granulosus also amplified DNA (117bp) from E. vogeli, and those designed for Taenia spp. amplified products (267bp) from species of Mesocestoides, Dipylidium and Diphyllobothrium. Nevertheless, as our diagnostic approach includes the concentration of taeniid eggs by sequential sieving and flotation, followed by their morphological detection, this non-specificity has limited practical importance. Sequence analysis of the corresponding amplicon can identify most of the described E. granulosus genotypes. Taenia spp. can be identified by direct sequencing of the 267bp amplicon, or, for most species, by restriction fragment length polymorphism (RFLP) analysis. The multiplex PCR was readily able to detect 1 egg (estimated to contain 7000 targets, as determined by quantitative PCR). Having been validated using a panel of well-defined samples from carnivores with known infection status, this approach proved to be useful for the identification of taeniid eggs from both individual animals and for epidemiological studie

An improved test system for PCR-based specific detection of Echinococcus multilocularis eggs

For the sensitive detection of eggs of Echinococcus multilocularis in fox faeces by PCR we have evaluated a method based on the previous concentration of helminth eggs by a combination of sequential sieving of faecal samples and flotation of the eggs in zinc chloride solution. The eggs were microscopically detected in the fractions retained in 40 and 20µm mesh sieves. DNA of the taeniid eggs retained in the 20 µm sieve was obtained after alkaline lysis and PCR was performed using E. multilocularis species-specific primers. Compared to the parasitological findings after examination of the small intestines of the foxes, the specificity of the PCR was 100% (no false-positive result with 20 foxes free of E. multilocularis) and the sensitivity was 94% (33 positive results from total 35 foxes proven to be infected with E. multilocularis). Both false-negative results were obtained with faeces from foxes harbouring immature worms. Using faecal volumes between 2 and 20 ml, no inhibition of PCR was observed as was demonstrated by the amplification of size-modified target in parallel reactions. The tests were undertaken with fresh faeces stored in 70% ethanol, but egg detection by PCR was also possible after inactivation of eggs by freezing the faeces at −80°C for one week or by incubation at +70°C for 2

Infectivity of Cryptosporidium parvum genotype I in conventionally reared piglets and lambs

Parasites of the genus Cryptosporidium are intracellular parasites that occur throughout the animal kingdom and have been reported in many species of mammals, including human. Most infections in humans are caused by two C. parvum genotypes, genotype I and genotype II; these are the human and the bovine (zoonotic) genotypes, respectively. Successful experimental infection of Cryptosporidium parvum genotype I "human genotype" is described in four conventionally reared piglets and in a lamb. The inoculum was originally obtained from two diarrheic children, and the Cryptosporidium genotypes were determined by PCR and rDNA sequencing. The infective dose was between 106 and 2×106oocysts. No clinical signs were observed in the infected animals, except in a piglet that showed watery diarrhea. The oocyst shedding period in positive animals ranged between 4 and 10 days. Histopathologic examination of the gastrointestinal tract of two positive piglets revealed shortening of the villi and denudation of the villous tips of the jejunum. In one piglet, the colon mucosa revealed numerous Cryptosporidium oocysts. The storage time of the inocula (≤3 weeks in PBS at 4°C) and the age of the animal (newborn) were important for the successful induction of infectio

Structural Characterization of a Cation-Selective, Self-Assembled Peptide Pore in Planar Phospholipid Bilayers.

GALA is a 30-residue amphipathic peptide that self-assembles into multimeric transmembrane pores in a pH-dependent fashion. In this study, we characterize the size, multimeric structure, and cation selectivity of GALA pores in planar phospholipid bilayers using electrical impedance spectroscopy and molecular dynamics simulations. We demonstrate that in planar bilayers GALA pores are likely formed by six peptide monomers rather than eight to 12 monomers as previously reported for lipid vesicles. We further show that in planar bilayers, GALA pores exhibit previously unreported cation selectivity. We propose that the difference between the predicted pore structures in planar bilayers and lipid vesicles exemplifies the importance of phospholipid bilayer structural properties on the aggregation of transmembrane helical structures

Age-dependent dynamics of Theileria equi and Babesia caballi infections in southwest Mongolia based on IFAT and/or PCR prevalence data from domestic horses and ticks

Epidemiological factors of tick-borne equine piroplasmoses, caused by Theileria equi and Babesia caballi, were investigated using logistic regression (GLM) and general additive models (GAM) based on the prevalences determined in 510 domestic horses and in ticks in S.W. Mongolia by indirect immunofluorescence antibody test (IFAT) and/or multiplex PCR. Prevalences of T. equi and B. caballi in horses were 66·5% (95% CI: 62·1-70·7) and 19·1% (15·6-22·9), respectively by PCR and 78·8% (74·9-82·3) and 65·7% (61·3-69·9) by IFAT. Of 166 ticks analysed from PCR- and IFAT-negative horses 1 was PCR positive for B. caballi and none for T. equi. GAM demonstrated non-linear increasing proportions of T. equi-PCR and -IFAT positive horses with age suggesting persistent infection. In contrast, the B. caballi-PCR prevalence decreased with age despite a concurrent increase in the proportion of IFAT-positive animals suggesting parasite elimination. The tick (Dermacentor nuttalli) burden of the horses increased with age and decreased with advancing season. Geldings were more likely to be infected with, and seroconvert to, T. equi. Neither herd affiliation, date of sample collection nor abundance of tick infestation had a significant influence on parasite prevalenc

Genetic characterization of Strongyloides spp. from captive, semi-captive and wild Bornean orangutans (Pongo pygmaeus) in Central and East Kalimantan, Borneo, Indonesia

Orangutans (Pongo spp.), Asia's only great apes, are threatened in their survival due to habitat loss, hunting and infections. Nematodes of the genus Strongyloides may represent a severe cause of death in wild and captive individuals. In order to better understand which Strongyloides species/subspecies infect orangutans under different conditions, larvae were isolated from fecal material collected in Indonesia from 9 captive, 2 semi-captive and 9 wild individuals, 18 captive groups of Bornean orangutans and from 1 human working with wild orangutans. Genotyping was done at the genomic rDNA locus (part of the 18S rRNA gene and internal transcribed spacer 1, ITS1) by sequencing amplicons. Thirty isolates, including the one from the human, could be identified as S. fuelleborni fuelleborni with 18S rRNA gene identities of 98·5-100%, with a corresponding published sequence. The ITS1 sequences could be determined for 17 of these isolates revealing a huge variability and 2 main clusters without obvious pattern with regard to attributes of the hosts. The ITS1 amplicons of 2 isolates were cloned and sequenced, revealing considerable variability indicative of mixed infections. One isolate from a captive individual was identified as S. stercoralis (18S rRNA) and showed 99% identity (ITS1) with S. stercoralis sequences from geographically distinct locations and host species. The findings are significant with regard to the zoonotic nature of these parasites and might contribute to the conservation of remaining orangutan population

Comparative copro-diagnosis of Echinococcus multilocularis in experimentally infected foxes

Faecal samples from 15 foxes experimentally infected with Echinococcus multilocularis were examined until 90days post-infection (dpi) by microscopical identification of eggs isolated by flotation/sieving, by coproantigen-enzyme-linked immunosorbent assay (cELISA), by polymerase chain reaction (PCR) on DNA, respectively, isolated directly from the faecal samples (copro-DNA PCR) and from the eggs obtained by the flotation/sieving procedure (egg-DNA PCR). Based on egg counts, three periods of the infection were defined: pre-patent (2-29dpi), high patent (30-70dpi) and low patent periods (71-90dpi). Whereas all methods were highly sensitive with samples from the high patent period, cELISA was the most sensitive to detect pre-patent infections (63%). Samples from the low patent infections were positive in 77% by microscopy and in 80% by egg-DNA PCR, being significantly more sensitive than cELISA and copro-DNA PCR. The isolation of eggs from the faecal material proved to be more sensitive by the flotation/sieving procedure as compared to the classical concentration McMaster techniqu

Amplification of cestode DNA from the peri-anal region of naturally infected foxes by PCR and LAMP: proof of concept for a potential sampling strategy for diagnosing human taeniosis

The diagnosis of human taeniosis can be achieved through coproscopy, ELISA or PCR. An important limitation of these methods is the high turnaround time for stool sample collection and preparation, indicating the need for a straightforward sampling strategy. Due to the high metabolic activity and reproductive potential of Taenia spp., we hypothesise that parasite DNA (cells and eggs) present in the peri-anal region of the host can be exploited as a target for molecular diagnosis. We evaluated the feasibility of recovering parasite DNA from the peri-anal area of foxes naturally infected with Taenia spp. Before necropsy, cotton swabs were rubbed at the peri-anal region of foxes. DNA was extracted using alkaline lysis coupled with a commercial DNA isolation kit (method A) or alkaline lysis alone (method B). DNA was used in the multiplex-PCR assay (previously described and called here swab-PCR) and a novel LAMP assay detecting Taenia spp. commonly found in foxes (swab-LAMP). The results of these assays from 105 foxes were compared with the presence of intestinal helminths determined at necropsy and by the sedimentation and counting technique (SCT). The sensitivity of swab-PCR for detecting Taenia (n = 68) was 89.8% (95% CI, 77.7–96.6) and 89.5% (66.9–98.7) using methods A and B, respectively. The sensitivity of the swab-LAMP assay was 83.7% (70.3–92.7) using method A and 89.5% (66.9–98.7) with method B. We postulate that peri-anal swab sampling followed by simplified DNA extraction and LAMP might be a suitable strategy for surveillance of human taeniosis in resource-limited settings in the future

Rodents as shared indicators for zoonotic parasites of carnivores in urban environments

Rodents are shared intermediate or paratenic hosts for Echinococcus multilocularis, Toxocara spp. and Toxoplasma gondii, and may serve as valuable indicators for assessing the occurrence and the level of environmental contamination and infection pressure with free-living stages of these zoonotic parasites. We investigated 658 non-commensal rodents for parasite infections in the canton of Geneva, Switzerland. The prevalence of infection with E. multilocularis was highest in Arvicola terrestris captured in the north-western area (16·5%, CI: 10·1%-24·8%), possibly reflecting a higher red fox density due to the low incidence of sarcoptic mange in this part of the canton. The exposure rate to Toxocara spp. was highest in the urban area (13·2%, CI: 7·9%-20·3%), and may account for higher densities of domestic carnivore and red fox definitive hosts within the city. Exposure to T. gondii was widespread (5·0%, CI: 3·2-7·4%), indicating a ubiquitous distribution of infected cat definitive hosts. Interestingly, a widespread distribution of Taenia taeniaeformis, a parasite mainly transmitted by cats, was similarly evidenced in A. terrestris. Distinct spatial patterns for the different zoonotic parasites likely reflected differences in distribution, abundance, and habitat use of the respective definitive hosts. These results highlight the potential value of rodents as shared indicators for these pathogen

Effects of alcohol consumption on mortality in patients with Type 2 diabetes mellitus

Aims/hypothesis: Moderate alcohol intake has been associated with increased life expectancy due to reduced mortality from cardiovascular disease. We prospectively examined the effects of alcohol consumption on mortality in Type 2 diabetic patients in Switzerland. Methods: A total of 287 patients with Type 2 diabetes mellitus (125 women, 162 men), recruited in Switzerland for the WHO Multinational Study of Vascular Disease in Diabetes, were included in this study. After a follow-up period of 12.6±0.6 years (means ± SD), mortality from CHD and from all causes was assessed. Results: During the follow-up, 70 deaths occurred (21 from CHD, 49 from other causes). Compared with non-drinkers, alcohol consumers who drank alcohol 1 to 15g, 16 to 30g and 30g or more per day had the following risk rates of death from CHD: 0.87 (95% CI: 0.25 to 2.51, NS), 0.00 (95% CI: 0.00 to 0.92, p less than 0.05) and 0.37 (95% CI, 0.01 to 2.42, NS), respectively. The corresponding risk rates of death from all causes were 1.27 (95% CI: 0.68 to 2.28, NS), 0.36 (95% CI: 0.09 to 0.99, p less than 0.05) and 1.66 (95% CI: 0.76 to 3.33, NS). Conclusions/interpretation: In Swiss Type 2 diabetic patients moderate alcohol consumption of 16 to 30g per day was associated with reduced mortality from CHD and from all causes. Alcohol intake above 30g per day was associated with a tendency towards increased all-cause mortalit