Prevalence and genotypes of Toxoplasma gondii in feline faeces (oocysts) and meat from sheep, cattle and pigs in Switzerland

The protozoan parasite Toxoplasma gondii infects almost all warm blooded animal species including humans, and is one of the most prevalent zoonotic parasites worldwide. Post-natal infection in humans is acquired through oral uptake of sporulated T. gondii oocysts or by ingestion of parasite tissue cysts upon consumption of raw or undercooked meat. This study was undertaken to determine the prevalence of oocyst-shedding by cats and to assess the level of infection with T. gondii in meat-producing animals in Switzerland via detection of genomic DNA (gDNA) in muscle samples. In total, 252 cats (44 stray cats, 171 pet cats, 37 cats with gastrointestinal disorders) were analysed coproscopically, and subsequently species-specific identification of T. gondii oocysts was achieved by Polymerase Chain Reaction (PCR). Furthermore, diaphragm samples of 270 domestic pigs (120 adults, 50 finishing, and 100 free-range animals), 150 wild boar, 250 sheep (150 adults and 100 lambs) and 406 cattle (47 calves, 129 heifers, 100 bulls, and 130 adult cows) were investigated by T. gondii-specific real-time PCR. For the first time in Switzerland, PCR-positive samples were subsequently genotyped using nine PCR-restriction fragment length polymorphism (PCR-RFLP) loci (SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) for analysis. Only one of the cats shed T. gondii oocysts, corresponding to a T. gondii prevalence of 0.4% (95% CI: 0.0-2.2%). In meat-producing animals, gDNA prevalence was lowest in wild boar (0.7%; 95% CI: 0.0-3.7%), followed by sheep (2.0%; 95% CI: 0.1-4.6%) and pigs (2.2%; 95% CI: 0.8-4.8%). The highest prevalence was found in cattle (4.7%; 95% CI: 2.8-7.2%), mainly due to the high prevalence of 29.8% in young calves. With regard to housing conditions, conventional fattening pigs and free-range pigs surprisingly exhibited the same prevalence (2.0%; 95% CI: 0.2-7.0%). Genotyping of oocysts shed by the cat showed T. gondii with clonal Type II alleles and the Apico I allele. T. gondii with clonal Type II alleles were also predominantly observed in sheep, while T. gondii with mixed or atypical allele combinations were very rare in sheep. In pigs and cattle however, genotyping of T. gondii was often incomplete. These findings suggested that cattle in Switzerland might be infected with Toxoplasma of the clonal Types I or III, atypical T. gondii or more than one clonal Type.

A nonsense mutation in the optic atrophy 3 gene (OPA3) causes dilated cardiomyopathy in Red Holstein cattle

Cardiomyopathies are severe degenerative disorders of the myocardium that lead to heart failure. During the last three decades bovine dilated cardiomyopathy (BDCMP) was observed worldwide in cattle of Holstein-Friesian origin. In the Swiss cattle population BDCMP affects Fleckvieh and Red Holstein breeds. The heart of affected animals is enlarged due to dilation of both ventricles. Clinical signs are caused by systolic dysfunction and affected individuals die as a result of severe heart insufficiency. BDCMP follows an autosomal recessive pattern of inheritance and the disease-causing locus was mapped to bovine chromosome 18 (BTA18). In the present study we describe the successful identification of the causative mutation in the OPA3 gene located on BTA18 that was previously reported to cause 3-methylglutaconic aciduria type III in Iraqi-Jewish patients. We demonstrated conclusive genetic and functional evidence that the nonsense mutation c.343C>T in the bovine OPA3 gene causes the late-onset dilated cardiomyopathy in Red Holstein cattle.

A longitudinal study of Besnoitia besnoiti infections and seasonal abundance of Stomoxys calcitrans in a dairy cattle farm of southwest France

Bovine besnoitiosis, caused by the cyst-forming apicomplexan Besnoitia besnoiti, is commonly reported in some restricted regions of South-Western Europe, and in larger regions of Africa and Asia. This infection is thought to be transmitted by blood feeding insects and is responsible for major economic losses in cattle production. A recent emergence in Europe, notified in the Centre of France, Spain and Germany, has attracted more attention to this disease. Clinical signs could appear in some animals; however, many infected cattle remain asymptomatic or show scleral-conjunctival cysts (SCC) only. Recent development of serological methods allows carrying out seroepidemiological field studies. In this respect, a long-term investigation was performed in a dairy cattle farm localized in an enzootic area of besnoitiosis of South-western France between March 2008 and May 2009. The objective was to estimate the seasonal pattern of B. besnoiti infections based on the presence of SCC and serology (ELISA and Western blot). In parallel, an entomological survey was conducted to describe population dynamics of Stomoxys calcitrans and Tabanidae species. The seroprevalence determined by Western blot in a cohort of 57 animals continuously present during the whole survey increased from 30% in March 2008 to 89.5% in May 2009 and was always higher than the prevalence based on clinically assessed SCC. New positive B. besnoitia seroconversions occurred throughout the year with the highest number in spring. In addition, many seroconversions were reported in the two months before turn-out and could be associated with a high indoors activity of S. calcitrans during this period.

Tritrichomonas foetus isolates from cats and cattle show minor genetic differences in unrelated loci ITS-2 and EF-1alpha

The protozoan parasite Tritrichomonas foetus is well known as an important causative agent of infertility and abortion in cattle (bovine trichomonosis). This World Organisation for Animal Health (O.I.E.) notifiable disease is thought to be under control in many countries including Switzerland. In recent studies, however, T. foetus has also been identified as an intestinal parasite that causes chronic large-bowel diarrhoea in cats. Since the feline isolates were considered indistinguishable from bovine isolates, the possibility and risk of parasite transmission from cats to cattle and vice versa has been intensively discussed in current literature. Therefore, we investigated if cat and cattle isolates are genetically distinct from each other or in fact represent identical genotypes. For this purpose, two independent genetic loci were selected that turned out to be well-suited for a PCR sequencing-based genotyping of trichomonad isolates: (i) previously published internal transcribed spacer region 2 (ITS-2) and (ii) a semi-conserved sequence stretch of the elongation factor-1 alpha (EF-1alpha) gene used for the first time in the present study. Respective comparative analyses revealed that both loci were sufficiently variable to allow unambiguous genetic discrimination between different trichomonad species. Comparison of both genetic loci confirmed that T. suis and T. mobilensis are phylogenetically very close to T. foetus. Moreover, these two genetic markers were suited to define host-specific genotypes of T. foetus. Both loci showed single base differences between cat and cattle isolates but showed full sequence identity within strains from either cat or cattle isolates. Furthermore, an additional PCR with a forward primer designed to specifically amplify the bovine sequence of EF-1alpha was able to discriminate bovine isolates of T. foetus from feline isolates and also from other trichomonads. The implications these minor genetic differences may have on the biological properties of the distinct isolates remain to be investigated.

Improved water vapour spectroscopy in the 4174-4300 cm-1 region and its impact on SCIAMACHY HDO/H2O measurements

The relative abundance of the heavy water isotopologue HDO provides a deeper insight into the atmospheric hydrological cycle. The SCanning Imaging Absorption spectroMeter for Atmospheric CartograpHY (SCIAMACHY) allows for global retrievals of the ratio HDO/H2O in the 2.3 micron wavelength range. However, the spectroscopy of water lines in this region remains a large source of uncertainty for these retrievals. We therefore evaluate and improve the water spectroscopy in the range 4174–4300 cm−1 and test if this reduces systematic uncertainties in the SCIAMACHY retrievals of HDO/H2O. We use a laboratory spectrum of water vapour to fit line intensity, air broadening and wavelength shift parameters. The improved spectroscopy is tested on a series of ground-based high resolution FTS spectra as well as on SCIAMACHY retrievals of H2O and the ratio HDO/H2O. We find that the improved spectroscopy leads to lower residuals in the FTS spectra compared to HITRAN 2008 and Jenouvrier et al. (2007) spectroscopy, and the retrievals become more robust against changes in the retrieval window. For both the FTS and SCIAMACHY measurements, the retrieved total H2O columns decrease by 2–4% and we find a negative shift of the HDO/H2O ratio, which for SCIAMACHY is partly compensated by changes in the retrieval setup and calibration software. The updated SCIAMACHY HDO/H2O product shows somewhat steeper latitudinal and temporal gradients and a steeper Rayleigh distillation curve, strengthening previous conclusions that current isotope-enabled general circulation models underestimate the variability in the near-surface HDO/H2O ratio.

Genetic analysis of syndactyly in German Holstein cattle

Congenital syndactyly with a variable number of affected feet was observed in eight black and white German Holstein calves. Analysis of the pedigree data revealed that all affected individuals could be traced back to a single founder. The pedigree was consistent with monogenic autosomal recessive inheritance and variable expressivity. Bovine syndactyly or "mulefoot" has been previously shown to map on the telomeric end of bovine chromosome 15 and we performed PCR genotyping of microsatellite markers spanning 27 cM of this chromosomal region to test the new cases for genetic linkage with the phenotype. The haplotype segregation confirmed the suggested inheritance pattern of the mulefoot mutation in this family and markers RM004, BM848 and BMS820 showed significant linkage to the phenotype. The results confirmed the chromosomal location of the mulefoot gene in this pedigree. Furthermore the study demonstrated that although marker testing has been available for nearly a decade the use of mulefoot carriers in cattle breeding remains uncontrolled. The presented family provides a resource for positional cloning of the causative mutation.

PRNP promoter polymorphisms are associated with BSE susceptibility in Swiss and German cattle

BACKGROUND: Non-synonymous polymorphisms within the prion protein gene (PRNP) influence the susceptibility and incubation time for transmissible spongiform encephalopathies (TSE) in some species such as sheep and humans. In cattle, none of the known polymorphisms within the PRNP coding region has a major influence on susceptibility to bovine spongiform encephalopathy (BSE). Recently, however, we demonstrated an association between susceptibility to BSE and a 23 bp insertion/deletion (indel) polymorphism and a 12 bp indel polymorphism within the putative PRNP promoter region using 43 German BSE cases and 48 German control cattle. The objective of this study was to extend this work by including a larger number of BSE cases and control cattle of German and Swiss origin. RESULTS: Allele, genotype and haplotype frequencies of the two indel polymorphisms were determined in 449 BSE cattle and 431 unaffected cattle from Switzerland and Germany including all 43 German BSE and 16 German control animals from the original study. When breeds with similar allele and genotype distributions were compared, the 23 bp indel polymorphism again showed a significant association with susceptibility to BSE. However, some additional breed-specific allele and genotype distributions were identified, mainly related to the Brown breeds. CONCLUSION: Our study corroborated earlier findings that polymorphisms in the PRNP promoter region have an influence on susceptibility to BSE. However, breed-specific differences exist that need to be accounted for when analyzing such data.

Local degradation of structural, mechanical, electrical and chemical properties of membrane electrode assembly in polymer electrolyte fuel cell

Polymer electrolyte fuel cell (PEMFC) is promising source of clean power in many applications ranging from portable electronics to automotive and land-based power generation. However, widespread commercialization of PEMFC is primarily challenged by degradation. The mechanisms of fuel cell degradation are not well understood. Even though the numbers of installed units around the world continue to increase and dominate the pre-markets, the present lifetime requirements for fuel cells cannot be guarantee, creating the need for a more comprehensive knowledge of material’s ageing mechanism. The objective of this project is to conduct experiments on membrane electrode assembly (MEA) components of PEMFC to study structural, mechanical, electrical and chemical changes during ageing and understanding failure/degradation mechanism. The first part of this project was devoted to surface roughness analysis on catalyst layer (CL) and gas diffusion layer (GDL) using surface mapping microscopy. This study was motivated by the need to have a quantitative understanding of the GDL and CL surface morphology at the submicron level to predict interfacial contact resistance. Nanoindentation studies using atomic force microscope (AFM) were introduced to investigate the effect of degradation on mechanical properties of CL. The elastic modulus was decreased by 45 % in end of life (EOL) CL as compare to beginning of life (BOL) CL. In another set of experiment, conductive AFM (cAFM) was used to probe the local electric current in CL. The conductivity drops by 62 % in EOL CL. The future task will include characterization of MEA degradation using Raman and Fourier transform infrared (FTIR) spectroscopy. Raman spectroscopy will help to detect degree of structural disorder in CL during degradation. FTIR will help to study the effect of CO in CL. XRD will be used to determine Pt particle size and its crystallinity. In-situ conductive AFM studies using electrochemical cell on CL to correlate its structure with oxygen reduction reaction (ORR) reactivity

Biochemical typing of pathological prion protein in aging cattle with BSE

BACKGROUND: The broad enforcement of active surveillance for bovine spongiform encephalopathy (BSE) in 2000 led to the discovery of previously unnoticed, atypical BSE phenotypes in aged cattle that differed from classical BSE (C-type) in biochemical properties of the pathological prion protein. Depending on the molecular mass and the degree of glycosylation of its proteinase K resistant core fragment (PrPres), mainly determined in samples derived from the medulla oblongata, these atypical cases are currently classified into low (L)-type or high (H)-type BSE. In the present study we address the question to what extent such atypical BSE cases are part of the BSE epidemic in Switzerland. RESULTS: To this end we analyzed the biochemical PrPres type by Western blot in a total of 33 BSE cases in cattle with a minimum age of eight years, targeting up to ten different brain regions. Our work confirmed H-type BSE in a zebu but classified all other cases as C-type BSE; indicating a very low incidence of H- and L-type BSE in Switzerland. It was documented for the first time that the biochemical PrPres type was consistent across different brain regions of aging animals with C-type and H-type BSE, i.e. independent of the neuroanatomical structure investigated. CONCLUSION: Taken together this study provides further characteristics of the BSE epidemic in Switzerland and generates new baseline data for the definition of C- and H-type BSE phenotypes, thereby underpinning the notion that they indeed represent distinct prion disease entities.

Intestinal Tritrichomonas foetus infection in cats in Switzerland detected by in vitro cultivation and PCR

Tritrichomonas foetus, a parasite well known for its significance as venereally transmitted pathogen in cattle, has recently been identified as a cause of chronic large-bowel diarrhea in domestic cats in the US, UK, and, more recently, also in Norway. In a period of 3 months (October to December 2007), 45 cats of Switzerland suffering from chronic diarrhea were investigated for intestinal infections, including a search for trichomonads. A commercially available in vitro culture system was used to screen for infection, complemented with a PCR and subsequent amplicon sequencing to support speciation. The PCR is based upon amplification of a sequence derived from the internal transcribed spacer region 1 (ITS1) on the ribosomal RNA gene (rRNA) using primers designed to detect a broad range of genera and species belonging to the family of Trichomonadidae. The method was furthermore adapted to the uracil DNA glycosylase (UDG) system in order to prevent carry-over contamination and it included a recombinant internal control to track for inhibitory reactions. Eleven out of the 45 cats were culture-positive, as revealed by microscopic identification of trichomonadid organisms. One of the isolates was subjected to scanning electron microscopy and findings revealed the presence of three flagella, thus placing the isolate into the gender Tritrichomonas sp. PCR and subsequent amplicon sequencing were carried out with ten of the 11 isolates. A total homology with published T. foetus sequences was confirmed in all of the cases. T. foetus therefore appears to range among those organisms that can cause chronic diarrhea in cats in Switzerland.

In Situ SHINERS at Electrochemical Single-Crystal Electrode/Electrolyte Interfaces: Tuning Preparation Strategies and Selected Applications

We have studied Au(55 nm)@SiO2 nanoparticles (NPs) on two low-index phases of gold and platinum single crystal electrodes in ClO4– and SO42– ion-containing electrolytes by both electrochemical methods and in-situ shell-isolated nanoparticle enhanced Raman spectroscopy (SHINERS). We showed the blocking of the electrode with surfactants originating from the synthesis of as-prepared SHINERS NPs. We introduce an efficient procedure to overcome this problem, which provides a fundamental platform for the application of SHINERS in surface electrochemistry and beyond. Our method is based on a hydrogen evolution treatment of the SHINERS-NP-modified single-crystal surfaces. The reliability of our preparation strategy is demonstrated in electrochemical SHINERS experiments on the potential-controlled adsorption and phase formation of pyridine on Au(hkl) and Pt(hkl). We obtained high-quality Raman spectra on these well-defined and structurally carefully characterized single-crystal surfaces. The analysis of the characteristic A1 vibrational modes revealed perfect agreement with the interpretation of single-crystal voltammetric and chronoamperometric experiments. Our study demonstrates that the SHINERS protocol developed in this work qualifies this Raman method as a pioneering approach with unique opportunities for in situ structure and reactivity studies at well-defined electrochemical solid/liquid interfaces.

Antibiotic Susceptibility of Mycoplasma bovis Strains Recovered From Mycoplasmal Pneumonia and Arthritis in Feedlot Cattle

Mycoplasmal pneumonia and arthritis is a problem of increasing significance in Midwestern feedlots. The disease presentation cannot be prevented by vaccination or successfully treated with antimicrobials. Due to the reported difficulty in treating these outbreaks, in-vitro antimicrobial susceptibility was tested on isolates of Mycoplasma bovis recovered from cases of pneumonia or pneumonia and arthritis where the mycoplasma was involved as a causative agent. Using a broth microdilution method, 36 M. bovis isolates from cases of pneumonia and 9 from cases of pneumonia and arthritis were tested for susceptibility to antimicrobials currently used in cattle with respiratory disease (ampicillin, tilmycosin, spectinomycin, tylosin, lincomycin, tetracycline, ceftiofur, and erythromycin). Among the isolates from cases with pneumonia, resistance to more antimicrobials was shown in recent isolates than in isolates from earlier years. Tetracycline and lincomycin were the drugs of choice for these isolates, although 3 of 36 isolates were resistant to all drugs tested. Isolates from cases of pneumonia and arthritis were from recent accessions. A majority of these isolates (5/9) were resistant to all antimicrobials tested. Lincomycin, spectinomycin, and tetracycline were antibiotics usable with 4/9 of the isolates. Overall, the results indicate that antimicrobial therapy in cases of mycoplasmal feedlot pneumonia and arthritis may be unrewarding.

Investigation of border disease and bovine virus diarrhoea in sheep from 76 mixed cattle and sheep farms in eastern Switzerland

The purpose of this study was to examine the occurrence of sheep persistently infected with Border disease virus (BDV) on 76 mixed cattle and sheep farms and whether seroconversion to BDV infection occurred in cattle of these farms. Seroprevalence of BDV and bovine viral disease virus (BVDV) infection in sheep was also investigated. Quantitative RT-PCR for pestivirus detection and an ELISA to detect pestivirus antibodies were used in 2'384 and 2'291 ovine blood samples, respectively. Another 27 seropositive sheep from ten flocks underwent serum neutralization testing to differentiate between BDV and BVDV antibodies. A BDV titre that was at least four times higher than the BVDV titre was interpreted as the result of BDV infection. Titres against BVDV were interpreted in an analogous fashion. All examined sheep were pestivirus-negative, 310 sheep were seropositive, 119 had an indeterminate titre and 1'862 were seronegative. The flock seroprevalence ranged from 0.0 to 73.9 %. Three of the 27 flocks that underwent serum neutralization testing were interpreted as BDV-infected because of 6 sheep with higher BDV titres, and 6 flocks were interpreted as BVDV-infected because of 14 sheep with higher BVDV titres.

Metamorphic and geochronogical study of the Triassic El Oro metamorphic complex, Ecuador: Implications for high-temperature metamorphism in a forearc zone

In the forearc of the Andean active margin in southwest Ecuador, the El Oro metamorphic complex exhibits a well exposed tilted forearc section partially migmatized. We used Raman spectroscopy on carbonaceous matter (RSCM) thermometry and pseudosections coupled with mineralogical and textural studies to constrain the pressure–temperature (P–T) evolution of the El Oro metamorphic complex during Triassic times. Our results show that anatexis of the continental crust occurred by white-mica and biotite dehydration melting along a 10 km thick crustal domain (from 4.5 to 8 kbar) with increasing temperature from 650 to 700 °C. In the biotite dehydration melting zone, temperature was buffered at 750–820 °C in a 5 km thick layer. The estimated average thermal gradient during peak metamorphism is of 30 °C/km within the migmatitic domain can be partitioned into two apparent gradients parts. The upper part from surface to 7 km depth records a 40–45 °C/km gradient. The lower part records a quasi-adiabatic geotherm with a 10 °C/km gradient consistent with an isothermal melting zone. Migmatites U–Th–Pb geochronology yielded zircon and monazite ages of 229.3 ± 2.1 Ma and 224.5 ± 2.3 Ma, respectively. This thermal event generated S-type magmatism (the Marcabeli granitoid) and was immediately followed by underplating of the high-pressure low-temperature (HP-LT) Arenillas–Panupalí unit at 225.8 ± 1.8 Ma. The association of high-temperature low-pressure (HT-LP) migmatites with HP-LT unit constitutes a new example of a paired metamorphic belt along the South American margin. We propose that in addition to crustal thinning, underplating of the Piedras gabbroic unit before 230 Ma provided the heat source necessary to foster crustal anatexis. Furthermore, its MORB signature shows that the asthenosphere was involved as the source of the heat anomaly. S-type felsic magmatism is widespread during this time and suggests that a large-scale thermal anomaly affected a large part of the South American margin during the late Triassic. We propose that crustal anatexis is related to an anomaly that arose during subduction of the Panthalassa ocean under the South American margin. Slab verticalization or slab break-off can be invoked as the origin of the upwelling of the asthenosphere.

Identification and quantification of phytochelatins in roots of rice to long-term exposure: evidence of individual role on arsenic accumulation and translocation

Rice has the predilection to take up arsenic in the form of methylated arsenic (o-As) and inorganic arsenic species (i-As). Plants defend themselves using i-As efflux systems and the production of phytochelatins (PCs) to complex i-As. Our study focused on the identification and quantification of phytochelatins by HPLC-ICP-MS/ESI-MS, relating them to the several variables linked to As exposure. GSH, 11 PCs, and As–PC complexes from the roots of six rice cultivars (Italica Carolina, Dom Sofid, 9524, Kitrana 508, YRL-1, and Lemont) exposed to low and high levels of i-As were compared with total, i-As, and o-As in roots, shoots, and grains. Only Dom Sofid, Kitrana 508, and 9524 were found to produce higher levels of PCs even when exposed to low levels of As. PCs were only correlated to i-As in the roots (r=0.884, P <0.001). However, significant negative correlations to As transfer factors (TF) roots–grains (r= –0.739, P <0.05) and shoots–grains (r= –0.541, P <0.05), suggested that these peptides help in trapping i-As but not o-As in the roots, reducing grains’ i-As. Italica Carolina reduced i-As in grains after high exposure, where some specific PCs had a special role in this reduction. In Lemont, exposure to elevated levels of i-As did not result in higher i-As levels in the grains and there were no significant increases in PCs or thiols. Finally, the high production of PCs in Kitrana 508 and Dom Sofid in response to high As treatment did not relate to a reduction of i-As in grains, suggesting that other mechanisms such as As–PC release and transport seems to be important in determining grain As in these cultivars.