Natural selection supports escape from concerted evolution of a recently duplicated CEACAM1 paralog in the ruminant CEA gene family

Concerted evolution is often observed in multigene families such as the CEA gene family. As a result, sequence similarity of paralogous genes is significantly higher than expected from their evolutionary distance. Gene conversion, a "copy paste" DNA repair mechanism that transfers sequences from one gene to another and homologous recombination are drivers of concerted evolution. Nevertheless, some gene family members escape concerted evolution and acquire sufficient sequence differences that orthologous genes can be assigned in descendant species. Reasons why some gene family members can escape while others are captured by concerted evolution are poorly understood. By analyzing the entire CEA gene family in cattle (Bos taurus) we identified a member (CEACAM32) that was created by gene duplication and cooption of a unique transmembrane domain exon in the most recent ancestor of ruminants. CEACAM32 shows a unique, testis-specific expression pattern. Phylogenetic analysis indicated that CEACAM32 is not involved in concerted evolution of CEACAM1 paralogs in ruminants. However, analysis of gene conversion events revealed that CEACAM32 is subject to gene conversion but remarkably, these events are found in the leader exon and intron sequences but not in exons coding for the Ig-like domains. These findings suggest that natural selection hinders gene conversion affecting protein sequences of the mature protein and thereby support escape of CEACAM32 from concerted evolution

Prevalência de anticorpos anti-Toxoplasma gondii e anti-Neospora caninum em suínos do Nordeste do Brasil

A serologic survey was conducted among 130 swine slaughtered in the public slaughterhouse of the city of Patos, Paraíba State, Northeastern Brazil, to determine the prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies, and to verify possible associations between sex of the animals and antibody prevalence. The sera were analyzed by indirect antibody tests, considering 1:64 (T. gondii) and 1:50 (N. caninum) dilutions as cut-off points. The prevalence of anti-T. gondii antibodies was 36.2% (47/130) (95% CI = 27.9 - 45.0%) with reciprocal titers ranging from 64 to 2,048, and of anti-N. caninum antibodies was 3.1% (4/130) (95% CI = 0.8 - 7.7%) with reciprocal titers ranging from 50 to 6,400. Three of the four N. caninum-positive samples were also positive for T. gondii antibodies. All Neospora and Toxoplasma IFAT-positive animals were also positive for confirmatory immunoblotting techniques using total and purified N. caninum and T. gondii tachyzoite antigens, i.e., p38 (NcSRS2) and p30 (TgSAG1). There was no association between sex of animals and prevalence of anti-T. gondii and anti-N. caninum antibodies. This is the first indication of N. caninum natural infection in pigs from Brazil.Foi conduzido um estudo sorológico em 130 suínos abatidos no matadouro público do município de Patos, Estado da Paraíba, Nordeste do Brasil, com o objetivo de determinar a prevalência de anticorpos anti-Toxoplasma gondii e anti-Neospora caninum, e verificar possíveis associações entre o sexo dos animais e a prevalência de anticorpos. Os soros foram analisados pelo testes de imunofluorescência indireta, considerando as diluições 1:64 (T. gondii) e 1:50 (N. caninum) como pontos de corte. A prevalência de anticorpos anti-T. gondii foi de 36,2% (47/130) (IC 95% = 27,9 - 45,0%) com títulos variando de 64 a 2.048, e anti-N. caninum de 3,1% (4/130) (IC 95% = 0,8 - 7,7%) com títulos variando de 50 a 6.400. Três das quatro amostras positivas para anticorpos anti-N. caninum também foram positivas para anticorpos anti-T. gondii. Todos os animais positivos na RIFI para Neospora e Toxoplasma também foram positivos nas técnicas confirmatórias de immunoblotting usando antígenos totais e purificados de taquizoítos de N. caninum e T. gondii, ou seja, p38 (NcSRS2) e p30 (TgSAG1). Não houve associação entre o sexo dos animais e a prevalência de anticorpos anti-T. gondii e anti-N. caninum. Essa é a primeira indicação de infecção natural por N. caninum em suínos do Brasil.Conselho Nacional de Pesquisa (CNPq

Analysis of Clonal Type-Specific Antibody Reactions in Toxoplasma gondii Seropositive Humans from Germany by Peptide-Microarray

BACKGROUND: Different clonal types of Toxoplasma gondii are thought to be associated with distinct clinical manifestations of infections. Serotyping is a novel technique which may allow to determine the clonal type of T. gondii humans are infected with and to extend typing studies to larger populations which include infected but non-diseased individuals. METHODOLOGY: A peptide-microarray test for T. gondii serotyping was established with 54 previously published synthetic peptides, which mimic clonal type-specific epitopes. The test was applied to human sera (n = 174) collected from individuals with an acute T. gondii infection (n = 21), a latent T. gondii infection (n = 53) and from T. gondii-seropositive forest workers (n = 100). FINDINGS: The majority (n = 124; 71%) of all T. gondii seropositive human sera showed reactions against synthetic peptides with sequences specific for clonal type II (type II peptides). Type I and type III peptides were recognized by 42% (n = 73) or 16% (n = 28) of the human sera, respectively, while type II-III, type I-III or type I-II peptides were recognized by 49% (n = 85), 36% (n = 62) or 14% (n = 25) of the sera, respectively. Highest reaction intensities were observed with synthetic peptides mimicking type II-specific epitopes. A proportion of the sera (n = 22; 13%) showed no reaction with type-specific peptides. Individuals with acute toxoplasmosis reacted with a statistically significantly higher number of peptides as compared to individuals with latent T. gondii infection or seropositive forest workers. CONCLUSIONS: Type II-specific reactions were overrepresented and higher in intensity in the study population, which was in accord with genotyping studies on T. gondii oocysts previously conducted in the same area. There were also individuals with type I- or type III-specific reactions. Well-characterized reference sera and further specific peptide markers are needed to establish and to perform future serotyping approaches with higher resolution

Peptide-microarray-based serological diagnosis and typing of Toxoplasma gondii infections in humans and cats from Germany

Mehrere Studien berichten über Hinweise, dass schwerwiegende Infektionsverläufe bei Menschen und Tieren mit bestimmten Genotypen und klonalen Linien von T. gondii in Verbindung stehen. Es ist daher wichtig, Kenntnisse über die Verbreitung unterschiedlicher Genotypen und Linien von T. gondii zu erlangen. Mithilfe dieser Kenntnisse könnten Infektionsgefährdungen für Menschen und Tiere besser abgeschätzt werden. Hinweise auf die Genotypen von T. gondii, mit denen Menschen oder Tiere infiziert sind, könnten in Zukunft frühzeitig Rückschlüsse auf den weiteren Infektionsverlauf liefern und gegebenenfalls eine Optimierung der Behandlung gestatten. Die Ergebnisse der in dieser Dissertation zusammengefassten Studien zeigen, dass Peptid- Mikroarrays geeignete Testsysteme für die serologische Diagnose und Typisierung von T. gondii-Infektionen bei Menschen und Katzen darstellen. Mit Hilfe von Peptid-Mikroarrays und Seren experimentell und natürlich infizierter Katzen konnte erstmals belegt werden, dass Katzen in der Lage sind, eine typspezifische IgGAntwort gegen T. gondii der klonalen Typen I, II, und III zu entwickeln. Nach Validierung wurden 27 Peptide selektiert, die für eine Serotypisierung der klonalen T. gondii-Infektionen verwendet wurden. Aufgrund früherer Studien konnte davon ausgegangen werden, dass die meisten Zwischenwirte in Deutschland gegenüber T. gondii des klonalen Typs II exponiert sind. Jedoch gab es keine Information zum Vorkommen unterschiedlicher T. gondii-Genotypen bei Menschen und nur im begrenzten Umfang Daten zum Vorkommen unterschiedlicher T. gondii-Typen bei Hauskatzen in Deutschland. Die vorliegende Arbeit zeigt, dass positive Typ-II-spezifische Peptidreaktionen bei serologisch positiven Menschen und Katzen signifikant überrepräsentiert sind. Die Reaktionen gegen Typ-II-spezifische Peptide waren signifikant stärker als die Reaktionen gegen Peptide anderer Spezifitäten. Die Ergebnisse weisen darauf hin, dass die meisten T. gondii-Infektionen bei Menschen und Hauskatzen in Deutschland durch T. gondii des klonalen Typs II verursacht werden. Die Studien bestätigen die Resultate vorheriger Arbeiten, in denen hauptsächlich Typ II bei den von Katzen in Deutschland ausgeschiedenen T. gondii-Oozysten nachgewiesen wurde. Da sporulierte Oozysten von T. gondii eine der wichtigsten Infektionsquellen für Zwischenwirte darstellen, war zu erwarten, dass es hinsichtlich der bei Katzen und Menschen beobachteten Serotypisierungs-Resultate keine wesentlichen Unterschiede geben sollte. Die hier durchgeführten Arbeiten repräsentieren eine erste Serotypisierung von T. gondii- Infektionen bei Menschen in Deutschland und eine erste bei Katzen weltweit mit Hilfe von Peptid-Mikroarrays. Allerdings zeigen die Ergebnisse, dass weitere gut validierte typspezifische Peptide erforderlich sind, um eine spezifischere Serotypisierung zu erreichen. Eine weitere Validierung mit einer größeren Zahl von gut definierten Seren könnte das Identifizieren und die Selektion polymorpher Peptide für die Serotypisierung erleichtern. Die in dieser Dissertation zusammengefassten Studien (Publikation 2, Manuskript 3) zeigen die Anwendbarkeit von bioinformatischen Verfahren wie „propensity scale“-Algorithmen und die ABCpred-Methode zur Vorhersage von linearen Epitopen in T. gondii-Antigenen. Mit der „propensity scale“-Methode wurden neun polymorphe Peptide ausgewählt, die als Teil von einem Peptid-Panel für T. gondii Serotypisierung eingesetzt werden könnten. Mit Hilfe von ABCpred konnten bioinformatisch insgesamt acht neue lineare Epitope vorhergesagt werden, die sich künftig auch als Teil eines diagnostischen Peptid-Panels einsetzen lassen. Insgesamt wurden in dieser Studie (Publikation 2) zehn Peptide (acht ABCpredvorhergesagte und zwei aus der Literatur) mit diagnostischem Potential identifiziert. Durch den Einsatz dieser Peptide im Peptid-Mikroarray zur serologischen Diagnose der humanen Toxoplasmose wurde eine diagnostische Sensitivität von 69% und Spezifität von 84% erreicht. Diese diagnostischen Parameter sind akzeptabel, aber nicht optimal. Um bessere diagnostische Eigenschaften des Testsystems erreichen zu können, ist es notwendig, das Peptid-Panel zu optimieren und nach weiteren Peptiden mit diagnostischem Potential zu forschen. Die Arbeit zeigt, dass die Anwendung bioinformatischer Programme zur Epitopvorhersage zusammen mit Peptid-Mikroarrays geeignete Laborwerkzeuge darstellen, mit denen T. gondii- Epitope identifiziert werden können, die sich zur serologischen Diagnose oder Typisierung eignen. Aus Peptiden, die diese Epitope enthalten, wurden Mikroarray-Tests entwickelt und zur serologischen Typisierung von T. gondii- Infektionen bei Menschen und Katzen eingesetzt.Several studies reported that certain T. gondii genotypes and clonal lineages correlate with severe toxoplasmosis in mice and humans. Therefore it may be important to have information on the prevalence of different T. gondii genotypes in humans and animals. In future, this information may help to better estimate the risk of infection for humans and animals. Information on the genotype of T. gondii an individual human or animal is infected may allow to conclude on the further development of disease and to optimise treatment. The results of studies summarized in this cumulative thesis demonstrate that a peptidemicroarray assay can be used for diagnosis of T. gondii infection and to detect T. gondii clonal type-specific antibody responses in seropositive humans and cats. With a peptide microarray and sera from experimentally- infected and naturally-infected cats it was shown for the first time that cats are able to mount a clonal type-specific IgG antibody response against T. gondii. After validation, 27 peptides were selected which were suitable for T. gondii serotyping in cats. Previous work suggested that individuals in the study area were mainly exposed to T. gondii clonal type II. However, there was no information, which clonal type of T. gondii is most prevalent in infected humans, and there was only limited data about the distribution of T. gondii types in cats in Germany. The results of this study demonstrate that positive peptide reactions presenting clonal type II were statistically significantly overrepresented in the tested human and cat population. The intensity, by which type II peptides were recognized, was also significantly higher than the intensity with which peptides of other specificities were detected. The results suggest that most T. gondii infections in humans and cats were caused by T. gondii clonal type II in Germany and confirmed previous findings reporting a pre-dominance of type II in oocysts shed by cats in Germany. Since sporulated T. gondii oocysts present one of the most important sources of infection for intermediate hosts, it was expected that there were no major differences in serotyping results between those for cats and humans. These are the first peptide microarray-based serotyping studies on T. gondii infections in humans in Germany and the first in cats worldwide. However, further carefully validated typespecific peptides are necessary to optimize the specificity of serotyping. Further validation with a larger number of well- characterized sera may help to identify and select further polymorphic peptides with a diagnostic potential. The results summarized in the present thesis (publication 2, manuscript 3) demonstrate that propensity scale-based and ABCpred bioinformatic methods are suitable for the in-silico prediction of linear epitopes on T. gondii antigens. The propensity scale method identified nine polymorphic epitopes that could be used as part of a peptide panel for T. gondii serotyping in cats. Using ABCpred, eight novel linear epitopes were predicted, which could be included in a larger peptide array for diagnosis. A total of ten peptides with diagnostic potential (eight ABCpred-predicted and two taken from the literature) were identified in this study (publication 2). The use of these peptides in a peptide-array revealed an overall diagnostic sensitivity of 69% and a diagnostic specificity of 84%. These diagnostic parameters are acceptable, but not optimal. To achieve better diagnostic properties, it is necessary to identify further epitopes with diagnostic potential to extend the peptide panel. In conclusion, this study demonstrates that bioinformatic programs in combination with peptide-microarray represent a powerful tool for the prediction and analysis of T. gondii linear epitopes suitable for serotyping or diagnosis. Based on peptides containing these epitopes peptide microarray tests were developed in the present thesis and applied or serological typing of T. gondii infections in humans and cats

Species Detection within the Echinococcus granulosus sensu lato Complex by Novel Probe-Based Real-Time PCRs.

Infections with eggs of Echinococcus granulosus sensu lato (s.l.) can cause cystic echinococcosis in intermediate host animals and humans. Upon ingestion of viable eggs, oncospheres hatch from the eggs and subsequently develop into fluid-filled larval cysts, most frequently in the liver or the lungs. The slowly growing cysts progressively interfere with organ function. The risk of infection is determined by the host range of the parasite, its pathogenicity and other epidemiologically relevant parameters, which differ significantly among the five species within the E. granulosus s.l. complex. It is therefore essential to diagnose the correct species within E. granulosus s.l. to help understand specific disease epidemiology and to facilitate effective implementation of control measures. For this purpose, simple, fast and cost-effective typing techniques are needed. We developed quantitative real-time polymerase chain reactions (qPCRs) to target polymorphic regions in the mitochondrial genome of E. granulosus s.l. In a single-step typing approach, we distinguished E. granulosus s.l. members in four epidemiologically relevant subgroups. These were E. granulosus sensu stricto, E. equinus, E. ortleppi and the E. canadensis cluster. The technique also allowed identification and differentiation of these species from other Echinococcus or Taenia taxa for samples isolated from cysts or faeces

Red foxes harbor two genetically distinct, spatially separated Echinococcus multilocularis clusters in Brandenburg, Germany.

BACKGROUND Alveolar echinococcosis (AE) is a clinically serious zoonosis caused by the fox tapeworm Echinococcus multilocularis. We studied the diversity and the distribution of genotypes of E. multilocularis isolated from foxes in Brandenburg, Germany, and in comparison to a hunting ground in North Rhine-Westphalia. METHODS Echinococcus multilocularis specimens from 101 foxes, 91 derived from Brandenburg and 10 derived from North Rhine-Westphalia, were examined. To detect potential mixed infections with different genotypes of E. multilocularis, five worms per fox were analyzed. For genotyping, three mitochondrial markers, namely cytochrome c oxidase subunit 1 (Cox1), NADH dehydrogenase subunit 1 (Nad1), and ATP synthase subunit 6 (ATP6), and the nuclear microsatellite marker EmsB were used. To identify nucleotide polymorphisms, the mitochondrial markers were sequenced and the data were compared, including with published sequences from other regions. EmsB fragment length profiles were determined and confirmed by Kohonen network analysis and grouping of Sammon's nonlinear mapping with k-means clustering. The spatial distribution of genotypes was analyzed by SaTScan for the EmsB profiles found in Brandenburg. RESULTS With both the mitochondrial makers and the EmsB microsatellite fragment length profile analyses, mixed infections with different E. multilocularis genotypes were detected in foxes from Brandenburg and North Rhine-Westphalia. Genotyping using the mitochondrial markers showed that the examined parasite specimens belong to the European haplotype of E. multilocularis, but a detailed spatial analysis was not possible due to the limited heterogeneity of these markers in the parasite population. Four (D, E, G, and H) out of the five EmsB profiles described in Europe so far were detected in the samples from Brandenburg and North Rhine-Westphalia. The EmsB profile G was the most common. A spatial cluster of the E. multilocularis genotype with the EmsB profile G was found in northeastern Brandenburg, and a cluster of profile D was found in southern parts of this state. CONCLUSIONS Genotyping of E. multilocularis showed that individual foxes may harbor different genotypes of the parasite. EmsB profiles allowed the identification of spatial clusters, which may help in understanding the distribution and spread of the infection in wildlife, and in relatively small endemic areas

Dog Ownership and Risk for Alveolar Echinococcosis, Germany

Human alveolar echinococcosis is caused by the parasite Echinococcus multilocularis, and dog ownership has been identified as a risk factor. We sought to specify the factors of dog ownership underlying this risk by conducting a case–control study among dog owners in Germany. The analysis revealed an increased odds ratio of ≈7-fold for dog owners whose dogs roam unattended in fields, 13-fold for dog owners who feed their dogs organic waste daily, 4-fold for dog owners who take their dog to a veterinarian only in case of illness, and 10-fold for dog owners who have never been informed by a veterinarian about the risk for infection. The results highlight the risk for infection associated with various factors of dog ownership and the value of veterinarians informing owners about prevention

Increasing Fatigue Life of 09Mn2Si Steel by Helical Rolling: Theoretical–Experimental Study on Governing Role of Grain Boundaries

The structure and mechanical properties of the 09Mn2Si high-strength low-alloyed steel after the five-stage helical rolling (HR) were studied. It was revealed that the fine-grained structure had been formed in the surface layer ≈ 1 mm deep as a result of severe plastic strains. In the lower layers, the “lamellar” structure had been formed, which consisted of thin elongated ferrite grains oriented in the HR direction. It was shown that the five-stage HR resulted in the increase in the steel fatigue life by more than 3.5 times under cyclic tension. The highest values of the number of cycles before failure were obtained for the samples cut from the bar core. It was demonstrated that the degree of the elastic energy dissipation in the steel samples under loading directly depended on the area of the grain boundaries as well as on the grain shapes. The fine-grained structure possessed the maximum value of the average torsional energy among all the studied samples, which caused the local material structure transformation and the decrease in the elastic energy level. This improved the crack resistance under the cyclic mechanical loading. The effect of the accumulation of the rotational strain modes at the grain boundaries was discovered, which caused the local structure transformation at the boundary zones. In the fine-grained structure, the formation of grain conglomerates was observed, which increased the values of the specific modulus of the moment of force. This could be mutually compensated due to the small sizes of grains. At the same time, the coarse-grained structures were characterized by the presence of the small number of grains with a high level of the moments of forces at their boundaries. They could result in trans-crystalline cracking

Effect of structural heterogeneity of 17Mn1Si steel on the temperature dependence of impact deformation and fracture

The paper deals with a theoretical and experimental study of the relationship between the microstructural parameters, mechanical properties, and impact deformation and fracture of steels using the example of 17Mn1Si pipe steel. A model for the behavior of a polycrystalline grain conglomerate under impact loading at different temperatures was proposed within a cellular automata framework. It was shown that the intensity of dissipation processes explicitly depends on temperature and these processes play an important role in stress relaxation at the boundaries of structural elements. The Experimental study reveals the relationship between pendulum impact test temperature and the deformation/fracture energy of the steel. The impact toughness was shown to decrease almost linearly with the decreasing test temperature, which agrees with the fractographic analysis data confirming the increase in the fraction of brittle fracture in this case. It was shown with the aid of the proposed model and numerical simulations that the use of the excitable cellular automata method and an explicit account of test temperature through the possibility of energy release at internal interfaces help to explain the experimentally observed features of impact failure at different temperatures

Increasing Fatigue Life of 09Mn2Si Steel by Helical Rolling: Theoretical–Experimental Study on Governing Role of Grain Boundaries

The structure and mechanical properties of the 09Mn2Si high-strength low-alloyed steel after the five-stage helical rolling (HR) were studied. It was revealed that the fine-grained structure had been formed in the surface layer ≈ 1 mm deep as a result of severe plastic strains. In the lower layers, the “lamellar” structure had been formed, which consisted of thin elongated ferrite grains oriented in the HR direction. It was shown that the five-stage HR resulted in the increase in the steel fatigue life by more than 3.5 times under cyclic tension. The highest values of the number of cycles before failure were obtained for the samples cut from the bar core. It was demonstrated that the degree of the elastic energy dissipation in the steel samples under loading directly depended on the area of the grain boundaries as well as on the grain shapes. The fine-grained structure possessed the maximum value of the average torsional energy among all the studied samples, which caused the local material structure transformation and the decrease in the elastic energy level. This improved the crack resistance under the cyclic mechanical loading. The effect of the accumulation of the rotational strain modes at the grain boundaries was discovered, which caused the local structure transformation at the boundary zones. In the fine-grained structure, the formation of grain conglomerates was observed, which increased the values of the specific modulus of the moment of force. This could be mutually compensated due to the small sizes of grains. At the same time, the coarse-grained structures were characterized by the presence of the small number of grains with a high level of the moments of forces at their boundaries. They could result in trans-crystalline cracking