[Rapid diagnosis of sexually transmitted infections : Joint statement of DSTIG, RKI, and PEI, as well as the reference centers for HIV, HBV, and HCV and consulting laboratories for Chlamydia, gonococci, and Treponema pallidum]. / Schnelltestdiagnostik sexuell übertragbarer Infektionen : Gemeinsame Stellungnahme von DSTIG, RKI, PEI sowie den Referenzzentren für HIV, HBV und HCV und Konsiliarlaboren für Chlamydien, Gonokokken und Treponema pallidum.

In February 2019, the fourth expert meeting on rapid diagnostic tests (RDTs) for sexually transmitted infections (STI) was held at the Robert Koch Institute (RKI) in Berlin. Novel technical developments and new aspects of RDT applications were discussed by representatives from the German STI Society (DSTIG); RKI; the Paul Ehrlich Institute; national reference centers for HIV, HBV, and HCV; and reference laboratories for Chlamydia, gonococci, and Treponema pallidum.As a result of this meeting, we present a revision of the joint statement on STI diagnostics with RDTs from 2017. The Regulation (EU) 2017/746 of the European Parliament and of the Council on in vitro diagnostic medical devices became effective in May 2017 and includes more stringent regulatory requirements for RDTs, mainly concerning conformity of manufacturing processes and performance characteristics of class D in vitro diagnostics (detection of HIV, HBV, HCV, and T. pallidum). Some RDTs for HIV, HCV, and T. pallidum have been evaluated in clinical studies and/or were WHO prequalified and may be used in low-threshold services. Among them are some HIV RDTs available and approved for self-testing. In addition, some HBV RDTs based on detection of HBs antigen (HBsAg) received WHO prequalification. However, false negative results may occur in samples with low HBsAg levels, as for instance in HIV-coinfected patients receiving antiretroviral therapy. For Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG), antigen-based RDTs still do not allow reliable detection of infection. Only PCR-based CT/NG RDTs possess sufficient diagnostic accuracy to be used as point-of-care tests. Rapid PCR tests for NG, however, do not provide any information about antimicrobial resistance.

Pilot study on binding of bovine salivary proteins to grit silicates and plant phytoliths.

Mostly fed with grass in fresh or conserved form, cattle and other livestock have to cope with silicate defence bodies from plants (phytoliths) and environmental silicates (grit), which abrade tooth enamel and could additionally interact with various salivary proteins. To detect potential candidates for silicate-binding proteins, bovine whole saliva was incubated with grass-derived phytoliths and silicates. Interactions of salivary proteins with pulverized bovine dental enamel and dentine were additionally analysed. After intense washing, the powder fractions were loaded onto 1D-polyacrylamide gels, most prominent adhesive protein bands were cut out and proteins were identified by mass spectrometry within three independent replicates. All materials were mainly bound by bovine odorant-binding protein, bovine salivary protein 30×10(3) and carbonic anhydrase VI. The phytolith/silicate fraction showed additional stronger interaction with haemoglobin ß and lactoperoxidase. Conceivably, the binding of these proteins to the surfaces may contribute to biological processes occurring on them.

Dietary abrasiveness is associated with variability of microwear and dental surface texture in rabbits.

Dental microwear and 3D surface texture analyses are useful in reconstructing herbivore diets, with scratches usually interpreted as indicators of grass dominated diets and pits as indicators of browse. We conducted feeding experiments with four groups of rabbits (Oryctolagus cuniculus) each fed a different uniform, pelleted diet (lucerne, lucerne & oats, grass & oats, grass). The lowest silica content was measured in the lucerne and the highest in the grass diet. After 25 weeks of exposure to the diets, dental castings were made of the rabbit's lower molars. Occlusal surfaces were then investigated using dental microwear and 3D areal surface texture analysis. In terms of traditional microwear, we found our hypothesis supported, as the grass group showed a high proportion of (long) "scratches" and the lucerne group a high proportion of "pits". Regardless of the uniform diets, variability of microwear and surface textures was higher when silica content was low. A high variability in microwear and texture analysis thus need not represent dietary diversity, but can also be related to a uniform, low-abrasion diet. The uniformity or variability of microwear/texture analysis results thus might represent varying degrees of abrasion and attrition rather than a variety of diet items per se.

Saliva proteomics as an emerging, non-invasive tool to study livestock physiology, nutrition and diseases.

Saliva is an extraordinary fluid in terms of research and diagnostic possibilities. Its composition in electrolytes, hormones and especially its proteome contains information about feeding status, nutritional requirements and adaptations to diet and environment, and also about health status of animals. It is easy to collect on a non-invasive and routine basis without any need for special training. Therefore, the analysis of salivary proteomes is going to emerge into a field of high interest with the future goal to maintain and improve livestock productivity and welfare. Moreover, the comprehensive analysis and identification of salivary proteins and peptides in whole and glandular saliva is a necessary pre-requisite to identify animal disease biomarkers and a powerful tool to better understand animal physiology. This review focuses on the different approaches used to study the salivary proteomes of farm animals, in respect to the physiology of nutrition and food perception in relation to food choices. The potential of animal saliva as a source of disease biomarkers will also be pointed out. Special emphasis is laid on the 'ruminating triad' - cattle, goat and sheep - as well as swine as major species of animal production in Western and Southern Europe.

First identification of tannin-binding proteins in saliva of Papio hamadryas using MS/MS mass spectrometry.

Hamadryas baboons possess salivary proline-rich proteins (PRP), as indicated by the presence of pink-staining protein bands using 1D SDS gel electrophoresis and Coomassie R250 staining. The ability of these protein bands to interact with tannic acid was further examined. In a tannin-binding assay using 5 µg tannic acid mixed with hamadryas whole saliva, we recently found four distinct protein bands of apparently 72, 55, 20, and 15 kDa that were precipitated during the experiments. In this work, we were able to identify these protein bands in a follow-up analysis using MS/MS mass spectrometry after excising such bands out of air-dried gels. Albumin and α-amylase were present in the tannic acid-protein complexes, with albumin already known to nonspecifically interact with a great diversity of chemical compounds. More interesting, we also identified a basic PRP and a cystatin precursor protein. This was the first successful attempt to identify a PRP from precipitated tannin-protein complexes in hamadryas baboons using MS/MS mass spectrometry. On the other hand, the role of cystatins in tannin binding is not yet well understood. However, there are recent reports on cystatin expression in saliva of rats responding to astringent dietary compounds. In conclusion, the follow-up data on tannin-binding proteins present in salivary secretions from hamadryas baboons adds important knowledge to primate physiology and feeding ecology, in order to shed light on the establishment and development of food adaptations in primates. It also demonstrates that tannin binding is characteristic for PRP, but might not be restricted to this particular group of proteins in primate species.

Morphological and physiological aspects of digestive processes in the graminivorous primate Theropithecus gelada-a preliminary study.

Hindgut fermentation has been suggested to contribute significantly to the digestive process in the gelada (Theropithecus gelada). We therefore hypothesized that in an in vitro fermentation test (Hohenheim gas test, using gas production as measure of microbial digestion) inoculum based on fresh gelada feces would degrade grass to a similar degree as zebra (Equus burchelli chapmani) feces and to a higher degree than that of hamadryas baboons (Papio hamadryas). Additionally, morphology of gelada tongue, salivary glands, stomach, and intestine were examined in this study. Gas production was measured between 4 and 96 hr using animal feces incubated with 200 mg of air-dry hay or mixed concentrate sample. For grass hay, 12-hr gas production was as follows: T. gelada (19.9 ml)>Papio (18.4 ml)>Equus (15.7 ml). After 24 hr, gas production changed: Papio (35.1 ml)>T. gelada (31.9 ml)>Equus (27.9 ml). At 96 hr, Papio was unexpectedly the most effective species with the highest gas production (53.1 ml)>zebra (51.2 ml)>gelada (49.4 ml). With a concentrate standard, 12-hr gas production was as follows: T. gelada (38.5 ml)>Equus (36.8 ml) = Papio (36.4 ml). At 24 hr, gas production differed: Papio (51.7 ml)>Equus (47.0 ml) = T. gelada (46.8 ml). At 96 hr, zebra was the most effective species with the highest gas production (63.9 ml)>Papio (60 ml) = T. gelada (59.9 ml). In conclusion, the results show that the microbial population present in gelada feces is able to ferment forage and concentrate substrates in vitro, although this fermentation did not occur with the expected effectiveness. Future studies should therefore focus also on the bacteria species involved.

Saliva of the graminivorous Theropithecus gelada lacks proline-rich proteins and tannin-binding capacity.

Gelada baboons are the sole survivors of the genus Theropithecus and the only known graminivorous primates. They developed special adaptations to their diet such as high-crowned teeth for processing hard and abrasive feed. The fine-tuning of salivary protein composition might be another key mechanism that is used by species for adapting to the environment and competing with rivals for exploiting new ecological niches. In order to test whether gelada (graminivorous) and hamadryas baboons (omnivorous) differ in their salivary protein composition, we compared whole saliva samples of captive Theropithecus gelada and Papio hamadryas using gel electrophoresis and tannin-binding assay. We hypothesized that the amount of proline-rich salivary proteins with tannin-binding capacity is higher in baboons consuming a feed with high dicot/monocot rations. Dicots produce tannins as a chemical defense system, discouraging animals from eating them. In contrast to dicots, monocots do not synthesize tannins. The presence of tannin-binding proteins in saliva should effectively inactivate the dicot tannin-based defense mechanism and increase the dietary breadth and/or the capability to switch between monocots and dicot leaves. The lack of such tannin-binding proteins in saliva would indicate a narrow dietary spectrum more restricted to monocots. We found T. gelada to completely lack proline-rich proteins (PRPs) and tannin-binding capacity similar to a great variety of other grazing mammals. In contrast, P. hamadryas does possess PRPs with tannin-binding activity. The findings support a growing body of evidence suggesting a high-level specialization of T. gelada to grass diets. However, it remains unclear, whether loss of salivary tannin-binding capacity drove the gelada into its narrow feeding niche, or whether this loss is the result of a long process of increased specialization. Thus, from an ecological point of view, T. gelada appears to be more vulnerable to environmental changes than other baboon species owing to its narrow dietary traits.

Evidence for the presence of carbonic anhydrase 29-kDa isoenzyme in salivary secretions of three ruminating species and the gelada baboon.

Salivary glands are highly variable in composition of their secretions and thus could be one of the primary ways by which species adapt or react to their environments. It has been hypothesized that feeding adaptation correlates with saliva composition. Hence, animals of different families using identical feeding niches should possess similar salivary proteins. For the first time, salivary secretions of grass-eating cattle, goat, camel and gelada baboon were compared by SDS-gel electrophoresis and immunoblotting. Salivary protein patterns were similar among individuals of the same species but varied largely among species. However, all samples showed proteins of apparently 29 and 42 kDa, identified as carbonic anhydrases (CA) by immunoblotting. The CA-VI (42 kDa) was highly expressed in cattle and camel saliva, but showed lower expression in goat saliva and could not be detected in gelada baboons. The CA-II (29 kDa) was found in saliva of all species tested and was shown in ruminating animals not to originate from cellular debris of the oral mucosa or ingested food. The results demonstrate that besides CA-VI, CA-II is another CA isoform secreted especially in ruminant saliva. Furthermore, the two CA isoenzymes detected may form a complementary system, protecting mucosa from acidity and helping to maintain a constant bicarbonate concentration in the animal's mouth and digestive tract.

Dose-dependent effects of genistein and daidzein on protein metabolism in porcine myotube cultures.

This study was conducted to investigate whether the isoflavones genistein and daidzein, which are components of soy-based diets, and the estrogen 17beta-estradiol affect differentiation and protein metabolism of porcine skeletal muscle cells in vitro. Serum-free porcine myotube cultures expressing the estrogen receptors ERalpha and ERbeta were treated with various concentrations of genistein, daidzein, or 17beta-estradiol for 26 h. The degree of differentiation by creatine phosphokinase activity was not altered by treatment. At 100 micromol/L both genistein and daidzein caused decreases in protein amount due to cell loss. In addition, 100 micromol/L genistein reduced protein synthesis rate of the surviving cells (P < 0.05) measured as [3H]-phenylalanine incorporation. Interestingly, genistein (0.1 micromol/L), daidzein (10, 100 micromol/L), and 17beta-estradiol (0.1, 1 nmol/L) slightly reduced protein degradation (P < 0.05). The results suggest that both genistein and daidzein affect protein metabolism in a dose-dependent manner and that estrogenic actions may play a role in decreasing protein degradation in porcine skeletal muscle.

IGF-I- and EGF-dependent DNA synthesis of porcine myoblasts is influenced by the dietary isoflavones genistein and daidzein.

Soy-derived isoflavones have been reported to be specific inhibitors of protein tyrosine kinases like the type 1 insulin-like growth factor receptor (IGF-1R) and the epidermal growth factor receptor (EGFR). This study was conducted to investigate, whether IGF-I and EGF stimulate porcine myoblast growth and whether the responses are influenced by isoflavones. Satellite cell-born myoblasts derived from the semimembranosus muscle of newborn piglets were treated for 26 h with IGF-I or EGF alone and in combination with genistein or daidzein. The DNA amount was measured and DNA synthesis was recorded as 6 h-[(3)H]thymidine incorporation during exponential growth in serum-free basal medium. IGF-I and EGF synergistically stimulated DNA synthesis of porcine myoblast with EGF causing a greater response. Genistein (100 micromol/l) effectively reduced the growth factor-mediated DNA synthesis, which was associated with an inhibition of growth factor receptor protein expression. In response to daidzein no reduction in growth factor-mediated DNA synthesis was found. Daidzein (1; 10 micromol/l) combined with IGF-I caused even a slight increase in DNA amount compared with the untreated control. The expression of the IGF-1R precursor protein was reduced with 10 and 100 micromol/l daidzein, whereas the EGFR expression remained unchanged with daidzein. The results suggest that dietary isoflavones may interact with growth factor-induced stimulation of pig skeletal muscle growth.

Developmental changes and the impact of isoflavones on mRNA expression of IGF-I receptor, EGF receptor and related growth factors in porcine skeletal muscle cell cultures.

OBJECTIVE: Soy that is widely used in human nutrition and in livestock production is a rich source of isoflavones. In addition to the estrogenic or antiestrogenic effects, isoflavones are suggested to affect cell growth via inhibition of protein tyrosine kinases (e.g. growth factor receptors). Therefore, the present in vitro-study was undertaken to determine, whether genistein and daidzein affect the mRNA expression of growth factor receptors (IGF-I receptor and EGF receptor) and their related growth factors in porcine skeletal muscle cell cultures. DESIGN: First, we investigated the basal mRNA expression of IGF-I, IGF-II, EGF, IGF-I receptor, and EGF receptor in proliferating and differentiating porcine skeletal muscle cell cultures using real-time PCR. Secondly, we measured the changes in the mRNA expression in these cell cultures treated with 0 (control), 1, 10, 100 microM genistein or daidzein over 26 h in serum-free medium (n=3). RESULTS: The mRNA expression of IGF-I was slightly decreased, whereas transcript concentrations of IGF-II and EGF were increased during differentiation compared with the proliferating stage of porcine muscle cell cultures. IGF-I receptor transcripts tended to be increased, whereas EGF receptor mRNA expression remained unchanged from proliferation to differentiation. Genistein and daidzein at 1 microM and 10 microM showed no effects on the mRNA expression of these genes, neither in proliferating nor in differentiating cells. However, high-concentrated isoflavones (100 microM) decreased the mRNA expression of IGF-I receptor and of the growth factors examined. CONCLUSIONS: The present study confirms the role of the IGF and EGF system in proliferation and differentiation of skeletal muscle cell culture especially under serum-free culture conditions. Furthermore, the results of this in vitro-study suggest that there is no effect of isoflavones at concentrations resulting from dietary consumption (1 and 10 microM) on IGF- and EGF-associated gene expression in porcine skeletal muscle tissue. Genistein and daidzein at high concentration (100 microM) reduced the mRNA expression of the IGF-I receptor and the growth factors examined, and therefore, may modify their autocrine and paracrine actions in skeletal muscle tissue.

Effects of dietary isoflavones on proliferation and DNA integrity of myoblasts derived from newborn piglets.

Soy-based formulas are consumed by growing numbers of infants and used as regular food supplements in livestock production. Moreover, constituent dietary phytoestrogens may compete with endogenous estrogens and affect individual growth. This study aimed to investigate the in vitro effects of isoflavones in comparison with estrogens on the proliferation of porcine satellite cells derived from neonatal muscle. After 7 h of exposure in serum-free medium, 17beta-estradiol (1 nM, 1 microM), estrone (1 microM), and daidzein (1, 100 microM) slightly decreased whereas 100 microM genistein substantially lowered DNA synthesis. Declines in DNA amount were observed with genistein (1, 100 microM) and daidzein (100 microM). After 26 h of exposure, 100 microM genistein reduced DNA synthesis, whereas it was increased by 10 microM genistein and 10 and 100 microM daidzein. In the case of 10 microM genistein and 100 microM daidzein, these increases apparently resulted from the repair of damaged DNA. Genistein and daidzein (100 microM) reduced protein synthesis, caused a G2/M phase block, and decreased DNA amount in association with higher rates of cell death partially resulting from apoptosis. Conclusively, isoflavones at concentrations of greater than 1 muM act as inhibitors of porcine skeletal muscle cell proliferation.

Establishment and conditions for growth and differentiation of a myoblast cell line derived from the semimembranosus muscle of newborn piglets.

To establish an adequate model to study the proliferation and differentiation of porcine skeletal muscle in response to bioactive compounds, a pool of satellite cells was derived from the semimembranosus muscle (SM) of newborn piglets using a Percoll gradient centrifugation. The final yield amounted to 4.1 x 10(6) cells/g muscle tissue. The percentage of muscle satellite cells has been determined by immunostaining for desmin and subsequent fluorescence analysis by flow cytometry, which revealed 95% of desmin-positive cells. For proliferation studies, satellite cell born myoblasts were seeded in gelatin-coated 96-well microplates at about 5 x 10(3) cells per well. Cells were grown for 1 day in MEMalpha plus 10% fetal bovine serum (FBS) and 10% horse serum (HS), followed by 2 d cultivation in serum-free growth medium. For differentiation studies, myoblasts were cultured in matrigel-coated 24-well plates for 4 d with growth medium containing 10% FBS and 10% HS. At 80% confluence, cells were grown for 24 h in medium plus 10% FBS and 1 microM insulin to initiate differentiation. Subsequently, the cells were cultured in serum-free differentiation medium (SFDM) for 3 d to form myotubes. Cultures reached a maximum fusion rate of approximately 20% after 96 h. By establishing this culture system, we provide an advanced and appropriate in vitro model to study porcine skeletal muscle cell growth and differentiation including the responses to various bioactive compounds.

Evidence for estrogen receptor alpha and beta expression in skeletal muscle of pigs.

Recent research suggests that estrogen receptors (ERs) are of significance in skeletal muscle function. The aim of the present study was to investigate, whether ERalpha and ERbeta are expressed in different porcine skeletal muscles and in satellite cells derived from semimembranosus muscle (SM) at the protein and mRNA level. Immunohistochemistry demonstrated positive staining for ERalpha in the nuclei of skeletal muscle cells, while the ERbeta stain showed positive signals in nuclei and cytoplasm of skeletal myofibers and myoblasts derived from satellite cells. Additionally, a weak expression of both ER subtypes was seen in skeletal muscle tissue and SM satellite cells with Western blot analysis. A clear expression of the ERalpha mRNA and a weak expression of the ERbeta mRNA was seen in skeletal muscle tissue and SM satellite cell cultures, as determined by reverse transcription (RT)-PCR. The present study shows for the first time that both ERalpha and ERbeta are expressed in porcine skeletal muscle, which, consequently, could be considered as a target tissue for estrogens or estrogen-like compounds. However, more detailed studies on the functional impact of both receptor subtypes in skeletal muscle are necessary. The porcine SM satellite cell culture provides a suitable in vitro model to investigate estrogenic effects on pig skeletal muscle.