Contagious agalactia (CA) of sheep and goats caused by Mycoplasma agalactiae is a widely occurring economically important disease that is difficult to control. The ELISA is commonly used for the serological detection of CA but it has some limitations and the performance of the available tests have not been properly evaluated.
ii. The influence of extrinsic factors such as the targeted animal species, geographical origin of the samples, intra-specific variability of M. agalactiae and concurrent mycoplasma infections.
A sample of 5900 animals from 211 farms with continuous CA monitoring for 20 years and no prior vaccination history was used. The infection status was known from prior bacteriological, epidemiological and serological monitoring with a complementary immunoblotting test.
The average diagnostic sensitivity was 56% [51.8–59.8] for the fusion protein ELISA and 84% [81.3–87.2] for the total antigen ELISA, with noteworthy flock-related variations. The average diagnostic specificity for the fusion protein ELISA was 100% [99.9–100], and for the total antigen ELISA differed significantly between goats and sheep: 99.3% [97.4–99.9] and 95.7% [93.8–97.2] respectively.
Experimental inoculations with different M. agalactiae strains revealed that the ELISA kits poorly detected the antibody response to certain strains. Furthermore, test performances varied according to the host species or geographical origin of the samples.
Finally, the correlation between milk shedding of M. agalactiae and the presence of detectable antibodies in the blood was poor.
These serological tests are not interchangeable. The choice of a test will depend on the objectives (early detection of infection or disease control program), on the prevalence of infection and the control protocol used. Given the variety of factors that may influence performance, a preliminary assessment of the test in a given situation is recommended prior to widespread use.
Contagious agalactia (CA) is a disease of sheep and goats mainly characterized by mastitis with a subsequent drop in milk production. Mastitis is often associated with arthritis and/or kerato-conjunctivitis and sometimes with pneumonia and septicemia in young animals. Different species of mycoplasma (bacteria lacking a cell wall, in the class Mollicutes) cause CA. The main species in both sheep and goats is Mycoplasma agalactiae and three other species produce a clinically similar disease in goats: M. capricolum subsp. capricolum, M. putrefaciens and mainly M. mycoides subsp. capri. M. agalactiae CA is found worldwide and is common in high milk-producing regions including countries bordering the Mediterranean sea . The welfare and especially economic consequences of this disease justify its inclusion in the list of animal diseases of global concern established by the World Organization for Animal Health (OIE) (http://www.OIE.int).
Until recently, M. agalactiae CA was enzootic in two French regions: the northern Alps in goats (Savoie and Haute-Savoie departments) and the western Pyrénées (Pyrénées-Atlantiques (P.A.) department) in dairy sheep . Mandatory screening based on serological ELISA tests was introduced into these areas 20 years ago. The disease appears to have been eradicated in the Alps as no M. agalactiae-infected herd has been identified for the last 10 years. In the P.A., M. agalactiae CA regularly regressed from 1993 to 2005 but then re-emerged abruptly. The number of outbreaks increased from 0 to 200 between 2006 and 2010, underlining the inadequacy or misapplication of the applied control methods .
Two methods are generally used for detecting M. agalactiae-infected farms (with or without clinical expression): indirect serological detection when animals are not vaccinated and direct evidence of the organism in milk (individual or bulk tank milks) . The two methods are complementary: a direct search in tank milk is useful for early detection of newly infected herds while serological testing can be used on non-milk producers or to detect animals with chronic or latent infection where little or no mycoplasma are shed in the milk .
Several serological ELISA tests for M. agalactiae have been described and compared (Table 1) [4–8]. They use as target antigen, either the total antigens of M. agalactiae strain(s), or fusion proteins, copies of M. agalactiae specific immunogenic proteins. According to the literature, the diagnostic specificity (percentage of uninfected animals that test negative) is between 76 and 99% for tests using total antigens and between 97 and 100% for tests using fusion protein(s). The diagnostic sensitivity (percentage of infected animals that test positive) varies widely between tests (48 to 94%) and also according to the study for a given test (56 to 82%) [6–8]. Such differences may be partly explained by inadequate sampling, the different geographical locations of the studies (New Zealand, Brazil, Italy, France), the animal species targeted (goats or sheep) or the infection stage.
(a) Formerly used commercial kit, (b) Test currently marketed and included in the study.
The objective of this study was to compare two ELISA tests that are widely used in Europe for the serological detection of M. agalactiae antibodies by evaluating: i) the performances per se : namely diagnostic sensitivity and specificity, the relevance of the recommended cut-off points, the correlation between the tests, and, the correlation between the presence of detectable antibodies and M. agalactiae shedding in milk; ii) the influence of extrinsic factors such as targeted animal species, geographical origin of samples, the intra-specific variability of M. agalactiae and mycoplasma co-infections.
The « POURQUIER-ELISA M. agalactiae » kit (IDEXX-Institut Pourquier, 326 rue de la Galera, F-36097 Montpellier cedex 5, France) is an indirect ELISA test. It targets antibodies against a fusion protein equivalent to M. agalactiae P48 protein  (P48-ELISA kit) and uses an anti-immunoglobulin G (IgG) conjugate. Normalised values (NV) based on optical density (OD) are given by the following formula: NV = (sample OD - negative control OD) × 100/(average OD of the positive control - average OD of the negative control). Results are interpreted as follows: negative when NV ≤ 50%, doubtful when NV is between [50-60%] and positive when NV ≥ 60%.
The « LSIVET M. agalactiae » kit (Laboratoire Service International, 6 allée des Ecureuils Parc Tertiaire Bois-Dieu, F-69380 Lissieu, France) is an indirect ELISA test that targets antibodies against the total antigens (TA-ELISA kit) of a M. agalactiae strain isolated from a goat in Spain. It uses an anti-IgG conjugate. Normalised values (NV) are given by the following formula: NV = sample OD/(2 x negative control OD). Results are interpreted as follows: negative when NV < 0.8, doubtful when NV is between [0.8-1] and positive when NV > 1.
The confidence interval for a single measurement was established from repeated measurements of a positive serum (PAL97) and three 2 by 2 dilutions of this serum on each test plate : 80 test plates of four different batches (20 plates by batch) for TA-ELISA kits and 60 test plates of two different batches (30 plates by batch) for P48-ELISA kits. The confidence interval on a single measurement was defined as +/− twice the standard deviation of the mean of the replicates . PAL97 serum was used as the positive reference serum for an European serological inter-laboratory testing (http://www.OIE.int). It originated from an outbreak of M. agalactiae CA in P.A. and was obtained from a ewe eight months after clinical recovery.
M. agalactiae detection in milk was performed in two steps. First, samples were enriched with mycoplasmas by two successive incubations at 37°C and 5%C02 for four days into appropriate broth media (Oxoid), secondly the presence of M. agalactiae was fast screened by a sensitive real time PCR test that targeted a M. agalactiae/M. bovis specific sequence located on the gene coding for 16 S RNA . Finally all cultures found positive or doubtful were tested again by a different PCR test that targeted the M. agalactiae specific P30 protein . Only positive samples with both PCR tests were considered as positive in the study.
Three hundred cultures that were positive with the above-described method were also tested by a dot-immunobinding technique .
Experimental infections were conducted with three different M. agalactiae strains: the PG2 reference strain, the 5632 strain isolated in Spain from a goat joint prior to 1991  and the 14628 strain isolated in France from bronchopneumonia lesions in a wild Capra ibex in 2006 . The Lyon Veterinary School Ethical Committee gave approval under agreement number #0730.
Test animals came from a goat herd that was certified free of pathogenic mycoplasma. Certification was based on regular bacteriological monitoring for the last five years (ear swabs, bulk tank and individual milk samples). All three experiments were performed under identical conditions: 2 to 3 adult goats were inoculated subcutaneously with 2 ml of culture containing 108–109 CFU/ml twice at a 16-day interval for strains 5632 and 14628 or at a 23-day interval for strain PG2.
Blood samples were taken every 3 days after the first inoculation until slaughter, 20 to 35 days later. The serological response was monitored by immunoblotting test and with both P48- and TA- ELISA kits.
Six sheep flocks infected by M. agalactiae (Table 2) were selected from the P.A. department, where CA has re-emerged recently. The bacteriological, serological and clinical statuses of the P.A. flocks were well known after 20 years of continuous CA monitoring without any vaccination scheme. The infected reference flocks were approximately the same size, and contained only one breed of sheep (Manech) reared under similar animal husbandry conditions. In particular, breeding was synchronized, so that milk production started at approximately the same time for all sheep and lasted 8 to 9 months. However the anteriority of M. agalactiae infection in each flock ranged from one to eight years after the first M. agalactiae detection in bulk tank milk. The prevalence of mastitis, arthritis and kerato-conjonctivitis during the study period was low in all flocks, less than 6%, but acute CA clinical signs reappeared during the following two years in 5 out of the 6 flocks. No animal purchase or grouping with another flock (mountain pasture, winter pension) occurred during the study.
(a) % of sero-positive animals in each flock.
(b) % of sero-positives in infected animals.
(c) Number of ewes that shed M. agalactiae (as determined by PCR testing of individual composite milk samples) at least once during four successive tests 5, 4, 3 and 1.5 months prior to blood sampling that took place 6 months after the beginning of lactation.
(d) Number of years between the present and first time M. agalactiae milk positivity was detected in the flock.
(e) Number of sero-positive animals in each flock.
(f) Exact binomial test distribution.
(g) Number of sero-positives in infected animals.
Infected reference animals were chosen from the six infected reference flocks described above. These flocks were monitored regularly during the 2009 milking campaign. For each lactating animal: i) milk samples (composite milk) were collected monthly from the second month after weaning and tested for the presence of M. agalactiae and ii) one blood sample was taken after six months of milk production.
Animals shedding M. agalactiae in milk at least once in the samples taken 150, 120, 90 or 45 days before blood sampling were classified as infected.
Four separate and independent animal populations were used in this study: two goat, one sheep and one mixed sheep/goat populations (Table 3).
M.m.capri = M. mycoides subsp.capri, CA: contagious agalactia.
Poitou-Charentes is the major area of goat milk production in France. The health status of the goat population is monitored regularly. Goats in this region are known to be frequently infected with M. mycoides subsp. capri but M. agalactiae is rare [15, 16]. Sixteen herds that had experienced an episode of clinical CA caused by M. mycoides subsp. capri (confirmed after isolation in tank and individual milk samples) were selected: 10 in 2003 and 6 in 2007. Approximately 10 individual serum samples were collected from each herd selected in 2003 and 30 samples in 2007. A total of 276 goat sera were analyzed.
The Haute-Savoie goat population has been under serological and bacteriological surveillance for M. agalactiae CA for over 15 years, and M. agalactiae infected herds have been systematically stamped out. No clinical outbreak of M. agalactiae CA has been detected since 2000. Sera collected during the 2007–2009 monitoring campaigns were used. 116 herds were selected at random: 87 contained fewer than 30 animals and 29 were large herds. All animals in the small herds were analyzed whereas only 20 randomly selected samples were analyzed in the large herds. In total, 1381 individual sera were analyzed.
The Tarn department is a major French producer of ewe's milk with no history of CA. Sera from 21 flocks averaging 400 heads were collected in 2007 during a Maedi-Visna surveillance campaign. Sera from 50 to 60 adult animals from each flock were analyzed, providing a total of 1195 sera.
Only the western part of the P.A. department (Basque country) is infected with M. agalactiae. The eastern part (Béarn region) has been disease-free for 10 years following an eradication program with stamping out of M. agalactiae infected flocks. Samples were analyzed from: i) 604 sheep from 43 randomly selected flocks, at a rate of about 15 adults from each flock (average flock size 300 sheep) and ii) all 275 adult goats from 9 herds.
M. agalactiae PG2 strain clone 55–5  was cultivated in liquid medium at 37°C for 48 hours. Cells were harvested by centrifugation at 12000 g for 20 min at 4°C followed by three washes in phosphate-buffered saline solution (PBS, 0.1 M Na2HPO4, 0.1 M Na2H2PO4, 0.15 M NaCl, pH 7.2). Washed cells were resuspended and stored at −20°C in the same buffer. The protein content of the cellular extract was determined as described by Lowry et al. . Samples were mixed with lysis buffer (0.5 M Tris/HCl pH 6.8, 4.6% (w/v) SDS, 20% (v/v) glycerol, 10% (v/v) 2-mercaptoethanol and 0.004% bromophenol blue) and boiled for 5 min. Proteins were separated on a 4-12% gradient Bis-Tris polyacrylamide gel (Invitrogen). An electrophoresis was run on the Invitrogen Xcell surelock system following the manufacturer's instructions. The separated proteins were transferred to a nitrocellulose membrane (Biorad) at 20 V constant voltage for 45 min in Tris glycine buffer (Tris 3.03 g/l, glycine 14.4 g/l) using the Biorad semi-dry transfer system. The membranes were blocked with a milk buffer (1 M glycine, 1% ovalbumin, 5% dry skim milk in PBS buffer) at room temperature and shaken for 2 hours then washed for 15 min at room temperature three times with PBS buffer 0.1% (v/v) tween 20 and finally with PBS alone. Each lane corresponding to a single well was cut, dried and immediately used or stored at −20°C. For immunoblotting, membranes were incubated for 2 hours at room temperature with 200 μl of serum diluted with 1 ml of dilution buffer (PBS containing 0.1% dry skim milk, 0.1% ovalbumin). After three washes in PBS 0.1% tween 20 and PBS buffer for 15 min at room temperature, the membranes were incubated for 2 hours at room temperature with an appropriate dilution of peroxidase-conjugated anti-sheep- or goat-IgG in dilution buffer then washed as described above. The peroxidase substrate containing 30 mg 4-chloro-1-naphthol dissolved in 10 ml methanol and 50 ml PBS and then 30 μl H2O2 was added and left in the dark for 5–15 min at room temperature.
A serum was considered positive if its profile contained four bands at 80, 48, 40 and 30 kDa simultaneously.
22 sera from infected reference ewes collected from the six infected reference flocks (Table 2). These animals shed M. agalactiae in milk and were positive with both P48 and TA-ELISA kits.
20 sera from ewes collected from flocks in disease-free areas and with no positive or doubtful ELISA result.
31 "doubtful" samples. These sera tested positive with the TA-ELISA kit. Animals originated from presumed M. agalactiae-free flocks that were thought to be free from M. agalactiae, based on bacteriological tank milk monitoring, but bordered the contaminated area of the Basque country.
When at least two animals from flocks located in CA-free areas tested positive with an ELISA test (2 to 12 per farm), the positive sera were re-assessed using IBT. A total of 20 sera from 5 flocks in the Béarn and 37 sera from 7 flocks in the Tarn were re-assessed.
Data management was conducted with Excel. The correlation between results from different tests was calculated using the Kendall rank correlation coefficient (or Kendall's tau coefficient) computed with R software (R Development Core Team, 2010, R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org/). The other statistical analyses were conducted using the EpiInfo® software. Confidence intervals of proportions were calculated using an exact binomial distribution. Comparisons between proportions were based on chi-square tests with an alpha level of significance of 5%.
All 22 sera from infected reference animals had an identical IBT profile characterized by simultaneous presence of four bands at 80, 48, 40 and 30 kDa and in most cases an additional band at 27 kDa (Figure 1-A). This profile was identical to the IBT profile obtained with sera from animals experimentally infected with the reference strain PG2 (Figure 2-A-a).
Immunoblotting profiles of sheep sera collected in the Pyrénées-Atlantiques department. A: sera from infected reference animals, B: sera from uninfected reference animals, C: sera from flocks thought to be uninfected but bordering newly infected flocks. Lane T: positive reference serum (PAL97). Black arrows indicate bands close to 80, 48, 40, 30 or 27 kDa. Total proteins from M. agalactiae PG2 reference strain clone 55.5  were used as antigen.
Kinetics of serological responses in goats inoculated experimentally with three strains of M. agalactiae. (A) Examples of immunoblotting obtained after inoculation: a) with PG2 reference strain; b) with 5632 strain; c) with14628 strain. Lane: 1 = J0, 2 = J + 6, 3 = J + 9, 4 = J + 12, 5 = J + 16, 6 = J + 19, 7 = J + 22, 8 = J + 30, days after first inoculation. Lane T: positive reference serum PAL97. Total proteins from the of M. agalactiae reference strain PG2 (clone 55–5)  were used as antigen. (B and C) Distribution of serum titers obtained respectively with TA-ELISA and with P48-ELISA. PG2: green curve, 5632: red curve, 14628: blue curve. Each curve corresponds to the average response of two animals for strains PG2 and 5632, three animals for strain 14628. Each point is an average of four repeated measures per animal. The manufacturer's suggested cut-off point is represented by the purple broken line. * Days of inoculation are symbolized with a star. J + x: number of days after the initial inoculation.
Several of the 20 sera from uninfected animals revealed IBT bands. However, the profiles were highly variable and never presented the 80-48-40-30 kDa bands simultaneously (Figure 1-B).
In general, the 31 doubtful samples had diverse profiles, all dissimilar to those of the infected reference animals. Only three samples (out of five) from one flock had profiles with bands at 80, 48, 40, 30 and 27 kDa (Figure 1-C). This farm experienced a clinical outbreak of CA in the following months.
The apparent prevalence of infection in the population of 2165 lactating ewes was calculated from the six infected reference flocks in the P.A. (Table 2). On average, 61% of the animals were positive with the TA-ELISA kit and 36% with the P48-ELISA kit. However, these averages masked differences between flocks. Major differences between the two tests were apparent in the two flocks that had been recently infected (one year ago). The correlation coefficient (Kendall's tau coefficient) between the two tests was 0.41 which is considered moderate.
Kit diagnostic sensitivity was estimated using sera collected from 609 infected reference ewes after six months of milk production (Table 2). The average diagnostic sensitivity of the TA-ELISA kit was 84% [81.3–87.2] and that of the P48-ELISA kit was 56% [51.8–59.8] (Table 2). The difference was significant (chi-square test, p < 10-6) indicating that the TA-ELISA kit was more sensitive. However, this average sensitivity masked major differences between flocks for both tests, ranging from 52% to 100% for the TA-ELISA kit and from 34 to 96% for the P48-ELISA kit. For the latter kit, the lowest values were obtained in recently infected herds.
Diagnostic specificity was estimated using 3711 sera from five distinct populations (Table 3). The specificity of the P48-ELISA kit was almost 100%, regardless of which M. agalactiae uninfected reference population was used. The specificity of the TA-ELISA kit varied from 95% to 100% according to the population and the animal species. Thus, 87 sheep in M. agalactiae-free populations (26 sheep from 14 flocks in the Béarn region and 61 sheep from 19 flocks in the Tarn department) gave positive results with the TA-ELISA kit. They were considered to be false positives as none of these sera had the IBT profile characteristic of M. agalactiae infection.
The confidence interval for a single measurement was approximately +/−20% regardless of the test, dilution of the reference serum or plate batch.
For both the TA- and P48-ELISA kits, the distribution of serum titers in four distinct sheep populations was expressed as the percentage of animals from a given population which fell into each titer category (Figures 3 and 4). For each kit, the titer distributions for two "M. agalactiae infected" and two "M. agalactiae uninfected" populations were plotted on the same graph. These were the two uninfected sheep populations from the Tarn department and Béarn region, the 609 infected reference sheep and finally a "probably infected" population consisting respectively of: i) 783 positive animals with the P48-ELISA kit for the titer distribution of the TA-ELISA kit and ii) 1090 positive animals with the TA-ELISA kit for the titer distribution of the P48-ELISA kit.
Distribution of serum titers obtained with TA-ELISA in four distinct sheep populations: - green curve: 1195 sheep from the M. agalactiae infection-free population from the Tarn department; - red curve: 604 sheep from the M. agalactiae infection-free population from the Béarn region;- blue curve: 609 ewes that shed M. agalactiae in at least one out of four successive individual milk analyses 5, 4, 3 and 1.5 months prior to blood sampling for serological testing; - black curve: 783 ewes from six infected flocks from the Pyrénées-Atlantiques department that tested positive with P48-ELISA. Titer distribution is expressed as the percentage of animals from a given population falling into in each titer category. The manufacturer's suggested cut-off point is represented by the purple broken line. The shaded area corresponds to a +/−20% confidence interval on a single measurement. The maximum titer frequencies for the Tarn and Béarn populations were respectively at 0.5 (32% of the population) and at 0.4 (25% of the population).
Distribution of serum titers obtained with P48-ELISA in four distinct sheep populations: - green curve: 1195 sheep from the M. agalactiae infection-free population from the Tarn department; - red curve: 604 sheep from the M. agalactiae infection-free population from the Béarn region; - blue curve: 609 ewes that shed M. agalactiae at least one out of four successive individual milk analyses 5, 4, 3 and 1.5 months prior to blood sampling for serological testing; - black curve: 1090 animals from six infected flocks from the Pyrénées-Atlantiques department that tested positive with TA-ELISA. Titer distribution is expressed as the percentage of animals from a given population falling into each titer category. The manufacturer's suggested cut-off point is represented by the purple broken line. The shaded area corresponds to a +/−20% confidence interval on a single measurement. The maximum titer frequencies for the Tarn and Béarn populations were respectively at 10% (97% of the population) and at 10% (57% of the population).
The positive cut-off point suggested by the TA-ELISA kit manufacturer (Figure 3) showed a good fit with the intersection of the infected and uninfected population distributions (with a confidence interval for a single measurement about 20%).
The cut-off point of 60% suggested by the P48-ELISA kit manufacturer (Figure 4) appeared excessive, given the distribution of the titers in both uninfected populations where no titer exceeded 25%. For a given 100% specificity and 20% confidence interval, lowering the threshold to 50% or 40% or 35% would increase the sensitivity of the test from 56% to 61%, 66% and 70% respectively (with a confidence interval for a single measurement about 20%).
The correlation between the presence of detectable antibodies in the blood and M. agalactiae milk shedding was poor for both tests with a Kendall correlation coefficient of 0.31 for the TA-ELISA kit and 0.26 for the P48-ELISA kit. Respectively 31% and 15% of the ewes that were known to have shed M. agalactiae between 45 and 150 days prior to blood collection tested negative with the P48-ELISA and TA-ELISA kits. This result was not attributed to false positive PCR reactions. The M. agalactiae detection was specific, the first positive PCR were confirmed by a second independent PCR. In addition 299 (99.7%) of the 300 cultures that tested M. agalactiae positive with PCR were also M. agalactiae positive when further tested by dot-immunobinding.
The serological responses to experimental infections with three M. agalactiae strains (PG2, 5632 and 14628) were monitored by IBT, and with the TA-ELISA and P48-ELISA kits (Figure 2).
IBT showed that all goats developed a significant humoral immune response following inoculation regardless of the strain used. No band was identified by IBT on the inoculation day, and multiple bands appeared from the 16th day after inoculation (Figure 2A).
Animals inoculated with strains 5632 and PG2 tested positive nine days later with the TA-ELISA kit and 16 days later with the P48-ELISA kit (Figures 2B and 2C). However the increase in antibodies of animals inoculated with strain 14628 was only weakly detected by the TA-ELISA kit and not at all with the P48-ELISA kit, despite a booster shot 16 days after the initial inoculation (Figures 2B and 2C).
In this study, the specificity of the TA-ELISA kit appeared to be lower in sheep (on average 95%) than in goats (99%). Specificity differed significantly between sheep and goats from the same geographic region (Béarn) (p = 0.005).
The specificity of the TA-ELISA kit differed significantly between three goat populations from three different geographical locations: Haute-Savoie, Poitou-Charentes and Béarn (p = 0.008). However, the specificity did not differ significantly between the two sheep populations from the Tarn department and Béarn region (p = 0.45).
In the 256 animals in the 16 herds currently infected with M. mycoides subsp. capri , only five in three herds were positive with the TA-ELISA kit, corresponding in 98% of specificity that was similar to that of other goat populations (Table 3).
This study compared two commercial ELISA kits widely used in Europe to detect antibodies against M. agalactiae, the primary agent of CA. Kits were tested on a large number of sera collected from 5900 animals from 211 farms. The constitution of representative samples of farms with well known infection status was possible for several reasons: first, the epidemiological situation of CA in France is well surveyed , second, a large number of farms and animals had been monitored for many years as part of several distinct eradication campaigns and third, animals had not been vaccinated against this agent. This large sample size guaranteed the statistical reliability and accuracy of the study. Nevertheless, the average performance values should be regarded with caution because a sampling bias may still have occurred, and furthermore, the values varied according to the region, host species or M. agalactiae strain.
Accurate determination of the infection status of individual animals regarding M. agalactiae is difficult in the absence of a gold standard diagnostic technique. Thus, the criteria used to define animals as "infected reference" differ from one study to another and may be clinical, bacteriological or serological [5–8]. The OIE recommends that in the absence of a gold standard for classifying an animal as infected, isolation of the disease agent can legitimately be considered the method of choice . Classification of an animal as infected may be biased because animals may shed the organism early in the course of infection even if the humoral immune response is still low. Furthermore, animals with latent infection that have developed an immune response may intermittently or no longer shed the organism and are therefore difficult to detect.
Here, the "infected reference" status of an animal was based on bacteriological identification of M. agalactiae in individual milk samples. The M. agalactiae detection protocol used in this study is highly sensitive and specific. Sensitivity was provided by the first step of culture: an inter-laboratory assay including 32 French diagnosis laboratories demonstrated that 70% of them detected M. agalactiae by culture when test aliquots contained only 2 to 25 CFU and 100% when test aliquots contained 25 to 250 CFU [unpublished data]. Specificity was provided by using two sets of PCR primers for the identification of M. agalactiae, and was confirmed by dot-immunobinding. Finally, bias associated with intermittent shedding and with early infection were limited by i) repeating bacteriological examinations of individual milk samples at regular intervals and ii) not taking into account milk samples performed during the period of 45 days prior to blood sampling.
The uninfected reference animals were recruited from geographical areas where M. agalactiae-related CA had never been reported or where routine monitoring had been in place for over 20 years and where no case had occurred in the last 10 years. Different independent populations and animal species (goats, sheep) were included to ensure diversity. Nevertheless, a number of animals, sometimes from the same farm, gave positive results with the TA-ELISA kit at the threshold recommended by the manufacturer. These positive results could have resulted from insufficient test specificity or from individual infections that escaped prior detection. A further technique was needed to unequivocally determine the status of these animals.
IBT is used for the serological diagnosis of contagious bovine pleuropneumonia (CBPP), another mycoplasma disease of ruminants, when routine tests are insufficiently accurate or ambiguous . Sera from infected animals give five specific IBT antigenic bands at 110, 98, 95, 60 and 48 kDa simultaneously (http://www.oie.int). IBT has been used in several studies to identify M. agalactiae infection in cases of CA [6, 7, 19, 20]. These authors described five stable immunogenic proteins with specific molecular masses of 80, 55, 48, 40, and 30 kDa, which presumably could be used as a specific signature of M. agalactiae infection much like with CBPP. However, three proteins (55, 30 and 40 kDa) are not expressed by all strains [6, 12, 21]. Thus an M. agalactiae-IBT should only be interpreted in the light of the antigenic profile of local strains.
A thorough analysis of 63 isolates collected between 1977 and 2007 in the P.A. showed that a single strain, very similar to the reference strain PG2, had been present for 30 years in the P.A . This strain expressed the 80, 48, 40, and 30 kDa proteins but not the 55 kDa one identified in Sardinian strains . A posteriori, the 55 kDa protein appeared to be antigenically similar to a variable protein of M. agalactiae known as Vpma U . On the PG2 antigenical phenotype used in IBT, Vpma U is apparent at 27 kDa. Understandably, if a single M. agalactiae strain, similar to PG2 reference strain, was circulating in the P.A., it seems reasonable to suspect that all serum samples from infected animals would express the same IBT profile with four major bands corresponding to stable proteins and a 27 kDa band, as confirmed by analysis of the 22 sera from infected reference animals. In contrast, the profiles of uninfected animals were heterogeneous and bands at 80, 48, 40, 30 kDa never occurred simultaneously. One recently infected flock that had escaped monitoring was detected by IBT. So IBT proved to be useful in determining the status of P.A. flocks with regard to M. agalactiae when the ELISA test results were ambiguous.
Several sera from the uninfected population gave positive results with the TA-ELISA kit. Even in the most doubtful cases (several positives in one farm), the IBT profiles were never similar to those of infected animals. Presumably, these were false positives and the wide distribution of non-negative responses to the TA-ELISA kit on several farms would support this hypothesis.
Variability within M. agalactiae strains can affect test performances, and test sensitivity may be improved by using a local strain as antigen . In the present study, the kinetics of antibody response were followed after experimental inoculations of goats with three different M. agalactiae strains. Detection of the immune response to one strain was poor with both ELISA kits. The variability of M. agalactiae is expressed in two ways. First, not all strains express certain specific immunogenic stable proteins [12–21]. Second, the expression and size of several surface proteins, such as the Vpma family, can show rapid and random fluctuations, thereby generating multiple antigenic surface configurations and broad intra-clonal variability . These two concomitant phenomena may alter the host's immune response, thereby affecting the sensitivity of diagnostic tests based on the degree of proximity between the phenotype of the test antigen and the phenotypes of the infecting strain. The consequences of antigenic variability on test performance are difficult to predict from strain characteristics. For example, both ELISA kits detect an immune response to strain 5632 even though this strain is genetically very different from the reference strain PG2 .
Test performance is also modified by geographic region and host species (sheep or goats). Regional differences may be due to antigenic variations between locally circulating strains. The TA-ELISA kit is less specific when used to detect M. agalactiae antibodies in sheep than in goats. This may be because it was developed with a goat strain that is possibly "adapted" to this species. Test specificity is not affected when goats are infected concurrently with other mycoplasma(s), even though these organisms may have many genes in common with M. agalactiae. However, given the genetic and antigenic variability of M. mycoides subsp. capri[15, 26], certain strains may well express atypical cross antigens, as is the case with CBPP .
The main source of CA contagion is by M. agalactiae shedding in milk [1, 2]. This study showed that the correlation between milk shedding and a detectable serological IgG response in blood was poor, implying that serology should not be used as an indicator of infectiousness. The percentage of animals that shed and tested negative with the TA-ELISA kit varied from 0 to 48% (average 16%) between farms, and from 4 to 62% (average 31%) with the P48-ELISA kit. Reports in the literature concerning the duration of the humoral response to mammary infection show considerable divergence, ranging from a rapid, systematic and persistent sero-conversion of at least 320 days  to a highly transient sero-conversion of less than 32 days . Further studies with reliable tests will be required to see whether the negativity in some infected animals results from the absence of serological response or from the rapid decrease of antibodies.
Two commercially available ELISA kits are widely used in several European countries to detect IgG antibodies against M. agalactiae.
The P48-ELISA kit is highly specific but not very sensitive. Its analytical performance characteristics may retard the detection of newly infected flocks especially when an insufficient number of animals are sampled. It is best used at the flock level.
The TA-ELISA kit is more sensitive, especially early in infection. On the other hand, it lacks specificity, especially with regard to sheep. When infection prevalence is low, false positive reactions hamper the monitoring and have to be tested again with expert serological test such as IBT. But no universal and standardized IBT test has been published for M. agalactiae yet.
In practice, the choice of kit should depend on: the user's objective (early detection of infection or ongoing control program), the epidemiological situation (prevalence) and the type of disease control program. The present ELISA kits because they do not detect all animals that are shedding M. agalactiae, are not well appropriate to estimate the infectious risk of a herd.
The analytical performance of both ELISA kits is highly subject to variations depending on the characteristics of the infecting strains. A preliminary assessment of their performance in a new situation is recommended prior to widespread use.
Future serological tests should involve several fusion proteins which reproduce antigens that are complementary, specific, stable and widely representative of M. agalactiae strains and furthermore are able to differentiate between responses to natural and vaccine antibodies.
The authors are grateful to 1) A. Paoli (Anses Lyon Laboratory) who carried out the immunoassays and was responsible for sera collection management, 2) The "Groupement de Défense Sanitaire" of P.A. in charge of the CA control plan, for providing us with sera and access to the flock sanitary status 3) P. Mercier (Anses Niort Laboratory) for providing us with goat sera.
FP conceived, coordinated and drafted the manuscript, DB coordinated all epidemiological and bacteriological studies in the P.A. and provided sera from the Tarn, MC and EG performed the statistical analysis, PG developed the immunoblotting test, YG coordinated serological monitoring in Haute-Savoie, DLG was in charge of experimental infections. All authors helped to improve the manuscript and approved the final version.


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Politics, The Left
UC-Berkeley Being Sued Over Coulter Speech
The College Republicans and the Young America's Foundation are suing the university saying they were, "seeking to "burden or ban" events "involving the expression of conservative viewpoints," according to Reuters.
By: Leesa K. Donner April 24, 2017 Politics, The Left
by Leesa K. Donner
If the freedom of speech may be taken away, then dumb and silent we may be led, like sheep to the slaughter. — George Washington
The leftist powers in charge at U.C. – Berkeley may have just overstepped their absurd bounds and caused even their political soulmates to call them on the carpet. Long heralded as the birthplace of the free speech movement, Berkeley has been the scene of violence when conservative provocateur Milo Yiannopoulous was sent packing instead of being permitted to speak on campus.
Now it seems they've stepped in it and drawn a lawsuit from the students who invited conservative firebrand Ann Coulter to speak in the hallowed halls of Berkeley. The College Republicans and the Young America's Foundation are suing the university saying they were, "seeking to "burden or ban" events "involving the expression of conservative viewpoints," according to Reuters.
The groups filed the lawsuit against Janet Napolitano, the president of the University of California, and other university officials in a San Francisco federal court on Monday. They asked the court to fine the school and award damages to organizers of the Coulter event.
Ms. Coulter says she intends to go forward with her talk on Thursday. Meanwhile, those on the left – yes, the left – are turning on Berkeley and standing with Ann:
From liberal TV host Bill Maher:
But Berkeley, you know, used to be the cradle of free speech, and now it's just the cradle for f—ing babies.
From Marc Randazza in an op-ed for CNN:
Berkeley should be the epicenter of the marketplace of ideas. Unfortunately, it has become the most intolerant place in America. I would feel more comfortable preaching for Sharia law in rural Mississippi than I would feel challenging the wage gap theory or speaking out against anti-Asian discrimination in admissions at Berkeley.
From Bernie Sanders:
To me, it's a sign of intellectual weakness," he said. "If you can't ask Ann Coulter in a polite way questions which expose the weakness of her arguments, if all you can do is boo, or shut her down, or prevent her from coming, what does that tell the world?
Instead of re-hashing all the many and various instances of conservatives being shut down on campuses nationwide, (most of which our readers already know) perhaps it's best to allow another famous – and hardly rightwing American have the last word to this latest First Amendment debacle in America.
Our lives begin to end the day we become silent about things that matter. — Martin Luther King, Jr.
Tags: 1st Amendment News Articles
Leesa K. Donner
Leesa K. Donner is Editor-in-Chief of LibertyNation.com. A widely published columnist, Leesa previously worked in the broadcast news industry as a television news anchor, reporter, and producer at NBC, CBS and Fox affiliates in Charlotte, Pittsburgh, and Washington, DC. She is the author of "Free At Last: A Life-Changing Journey through the Gospel of Luke."
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redpajama_set_name: RedPajamaCommonCrawl


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There's nothing like having a common cold to bring out the voodoo in one's friends. As I write this I'm a week into a cold, the first for well over a year. I've had a bad sore throat, aches and pains, and now I'm starting to get swollen sinuses and excessive mucus production. Classic viral infection. It's a story that must have been playing out in humans for 10's of 1000s of years. Apparently there are over 200 variations of the viruses that cause the range of symptoms that we call "a cold". We call it a "cold" or a "chill" because historically we thought that cold damp air upset the four bodily humours, and caused the symptoms. Some people still think that being cold and damp causes colds, though this is not true.
One of the main recommended therapies, as I'm sure readers will know, is vitamin C and lots of it. Tell someone you have a cold and like a Jehovah Witness on the doorstep asking if you "know Jesus", they will ask you if you're taking Vitamin C, because "It helps me so much when I have a cold". Hallelujah!
We have Nobel Laureate Linus Pauling to thank for popularising Vitamin C. Back in 1970 he published a book called "Vitamin C and the Common Cold" which made some rather bold claims. Unfortunately the studies he based his book on have subsequently been discredited (by one of the authors of the study cited below). However he stimulated interest and since then many trials of varying have been conducted, but usually with confusing results. The way to deal with confusing results to do a review: combine all the studies done over a period and see what the averages look like. One of the largest ever reviews of research on Vitamin C was published in 2013.
"... regular supplementation had a modest but consistent effect in reducing the duration of common cold symptoms, which is based on 31 study comparisons with 9745 common cold episodes."
"Trials of high doses [in the order of 8000mg] of vitamin C administered therapeutically, starting after the onset of symptoms, showed no consistent effect on the duration or severity of common cold symptoms."
The regular supplement was between 200 mg and 2000 mg with the most common dose being 1000 mg. The mean reduction in the duration of the cold symptoms for adults was 7.7% (ranging between 4 and 12%). Thus if my present cold lasts 14 days (which seems to be normal for me in the UK) I could expect a reduction in the duration of my symptoms of approximately one day from taking 1000 mg of vitamin C per day. Taking doses of 8000 mg per day showed no increased benefit unless one was a marathon runner (or similar) in which case it was associated with a moderately lower the risk of infection. One day out of 14 is hardly spectacular and is certainly well under the perceived benefit that people claim to gain from taking it. Mind you the study showed considerable variation. It might be almost twice as much (almost 2 days out of 14), but it might be half as much.
Something else to keep in mind is that with 200 separate viral agents causing a wide range of symptoms there is no standardised time that a cold lasts. Two people could even be infected with the same virus, at the same time, and still have symptoms of varying duration because of factors unknown. Of course scientists use control groups and statistical analysis to try to determine what is significant, but when every case of the common cold is different it's important not to overstate the value of these studies. For a lay person, dosing themselves with store-bought vitamin C and no control group, its not really possible to make sensible deductions about the efficacy of the stuff on their illness. Most of the studies that have been conducted have in fact been inconclusive or contradictory. Many of the studies conducted in the immediate aftermath of Pauling's book found no evidence at all for therapeutic effects of vitamin C. It's only in a large review that some clarity emerges.
So if the measurable effect is roughly one day in 14 why do people absolutely swear by Vitamin C and hawk it like bible-thumping evangelists? As pointed out in my précis of work on reason by Mercier & Sperber, we humans are very poor at solo reasoning tasks. Reason is a process that seems to have evolved for small groups to make collective decisions. One person proposes as idea and presents all the evidence for it (and thus confirmation bias is a feature of reasoning, not a bug), while other group members critique the idea and/or propose alternatives. Reasoning helps lead a small group to a decision. It does not necessarily lead to the truth or the Truth, but simply helps a small group decide what actions to take. Presumably the results of previous decisions play a part in critiquing the idea, but most of us simply cannot both propose and idea and critique it at the same time. Thus it's natural to argue for a remedy like vitamin C if one believes in it and to martial all the facts at one's disposal to support the argument. It's just that people making arguments find it hard to look beyond facts that confirm there argument. And it's up the rest of the group to come up with counter arguments. When a meme like "vitamin C is good for colds" becomes popular (due to a Noble laureate describing it as a panacea) then there are few people making the counter-argument and the reasoning process simply fails to work. There is no one to play the Devil's advocate and without that a theory cannot be properly thought about, let alone tested.
There is another possibility. I don't have my copy to hand, but there is an interesting supplement in Ariel Glucklich's book The End of Magic to do with perceptions of time. While studying tantric healers in Varanasi, he also came to know some divers. These men free dived from rowing boats to find treasure on the bottom of the Ganges (I think it might have been gold from corpses, but the reason is immaterial). Asked how long they could hold their breath these men answered in all sincerity "15-20 minutes". Which is incredible! Or it would be if it were true. In fact when timed they were under for about 5 minutes, which is about the physiological limit and similar to pearl divers and others to do this for a living. So where they lying? Glucklich concluded that they were not lying, but their sense of time when underwater became distorted. The diving is in fact quite dangerous as the river has strong currents and a huge amount of debris. Diving stretches the body's physiology, but when you add danger it magnifies the effect. Subjectively the divers experienced the time as much longer than the more objective observer sitting safely on the boat with a stop-watch.
I wonder if something similar happens to the ill person? Being ill seems interminable. I for one sleep badly when I have a cold and feel wretched for days on end primarily because of this. Perhaps time perception is altered. And something which promises to reduce the time of suffering becomes subjectively inflated. I wonder if an effect like this could be measured?
Children are more responsive to the vitamin than adults"
Remembering that the "exercise" referred to was marathon running and the associated doses of vitamin C were in the order of 8000 mg which is 10,000 times the daily requirement for the stuff in the body. The result doesn't apply to the kind of moderate exercise that most people manage. In fact they chose not to focus on the result in the way that I have, i.e. on the demonstrable (if variable) therapeutic effectiveness of vitamin C in reducing the duration of symptoms. That was a more positive story. The Mail, generally speaking, is a right-wing paper with an agenda that includes demonising the poor and unhealthy. It is famous for regularly featuring front page stories on the latest cause of cancer or cure for cancer, with a preference for the former. In the Mail's world everything is either a cause or cure of cancer. The slightest hint that a substance, or especially a food, causes cancer and it goes on the front page. So for them it's natural to see the story as a criticism of "couch potatoes" and to focus on the negative side of health stories.
G. Michael Allan and Bruce Arroll. 'Prevention and treatment of the common cold: making sense of the evidence.' CMAJ (published ahead of print January 27, 2014).
But this is not available for free online reading (unlike the previous study). So the average person simply hits a dead end at this point. There is no way to check what the journalist is saying, and in the case of the Telegraph the journalist has clearly mislead the reader. If you search for research on this subject the results vary so wildly that it is very confusing. How is a lay person supposed to make an informed decision? The simple answer is that we don't. We almost never do. We decide based on what we already know, what our friends and family say, and some fairly random research on Google. Most of us are not able to assess the reliability of the information. It's an invidious position to be in.
Happily the large meta-analysis is free online, and these large studies of studies are probably the best way to assess the effectiveness of any given treatment. So we can rest assured that in the case of vitamin C it's worth taking when you have a cold.
I thought of this point having more or less finished the essay above. It might have been possible to weave into the main narrative, but in a way it works as a standalone comment because it highlights something about the public perception of medicines. Notwithstanding the poor standards of science reporting in the newspapers, the studies referred to above are very careful to eliminate the placebo effect by using double blinds and randomisation, so that even those administering the treatment don't know who is being given what. The placebo effect was first noticed because it skewed results in drug trials where the participants or the doctors knew what treatment was being given. When the patient believes that they are receiving an effective treatment that treatment is more likely to be effective and/or like to be more effective than if they believe they are getting no treatment (there's some suggestion that the knowledge of the administering doctor can also have an effect). From the scientists point of view this means they can be fooled into thinking that a drug is more effective than it is (they are really only interested in the chemistry and not the psychology of drugs they hand out).
Vitamin C + credulity with respect to vitamin C are (on average) more effective than vitamin C alone.
Of course no one measures this extra effectiveness, because the scientific credibility of drugs rests on their their chemical effects. But where it has been measured, the placebo effect is really quite a significant factor in health treatments.
Lay people might like to think of placebo as the credulity bonus. Doctors of course are supposed to be forbidden by moral codes from over-stating the effectiveness of drugs, despite the fact that many drugs -- like antibiotics and antidepressants -- are routinely over-prescribed. It would certainly be less harmful to prescribe vitamin C for colds than it is prescribing antibiotics. The excess vitamin C over the ~40mg or so recommended by the NHS is just excreted in urine and there are few side-effects even at relatively high doses.
next week it's back to limbo.


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Vehicles | April 6, 2017 9:00 am
The Panthermobile Was Made for One Thing and One Thing Only
Wet bar. Pink shag carpet. Bow chika wow wow.
By Evan Bleier @itishowitis
No offense the original Batmobile, but if we're picking our favorite car from a painfully dated television program, we're taking the uber-groovy Panthermobile.
Designed as the flagship vehicle for The Pink Panther Show, which first aired in 1969, the Panthermobile is a 23-foot-long custom limo that's built on a '60s Oldsmobile Toronado chassis. It is the ultimate slice of gaudy '70s kitsch, and we love it.
Safety was not the main concern during the crafting of the front-wheel-drive car: the driver's seat is located outside the car directly in front of the vehicle's seven-liter engine. However, the placement of the cockpit does allow the occupants of the thin-snouted car to enjoy some privacy.
Given the miniature wet bar and the full-on pink shag carpet, privacy is probably something you'd want if joyriding around in this whip.
Additional features on the car — which cost $100,000 to build and was sold at auction for substantially more than that in 2011 following a full restoration by Galpin Auto Sports — include two-piece clamshell doors, a backup camera linked to a small TV and a vintage '70s car phone.
If you're a panther looking for a cougar, you could do a lot worse than this. Check out more looks in the full gallery below.
Panther (4 images)
Last Night's "SNL" Featured the "Parks and Recreation" Crossover You Never Expected
Bill Maher Returns With a New "Real Time" Season, Green Day Theme Song and All
LIV Golf Set to Announce TV Deal With The CW Network?


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Click to enquire , or call: 020-3642-4246 | 1-844-677-7010
Villa Pardo – Sustainable rural tourism in Buenos Aires Province
by Rebecca
agriculture, bird-watching, Buenos Aires, camping, colonial architecture, cultural heritage, cycling, organic farming, rural tourism, social and environmental projects, sustainable development, traditional gastronomy
Villa Pardo is much more than a rural tourism destination; it is a small town that has harnessed its history, identity and natural heritage to look towards the future. Located just 220km from the Argentine capital, members of the community have come together to reverse the town's decline and champion a kind of tourism firmly rooted in the principles of sustainable development. Today, Villa Pardo offers travellers the chance to experience a new and forward-thinking form of rural tourism in Buenos Aires province.
Villa Pardo is one of the many towns that sprung up alongside the railway tracks of the Buenos Aires Great Southern Railway in the second half of the nineteenth century. Once described as "one of the best towns in the world" by its most famous son, multi-award-winning author Adolfo Bioy Casares, the town has since suffered from a decline in fortune. A fate shared with many provincial towns, Villa Pardo has seen its population dwindle due to a lack of local opportunities and youth leaving in favour of bigger towns and cities.
Rural tourism and sustainable living
Faced with these challenges, members of the community got together with the aim of instilling a new lease of life into the town. They settled on the twin concepts of 'rural tourism' and 'sustainable living' and have built an innovative and exciting tourism offering around these pillars. Central to their objectives are the provision of local employment and the revitalisation of local identity and traditions.
Attending a sustainability workshop in Villa Pardo, Buenos Aires, Argentina
The aim was never for the town to reinvent itself, but for it to harness its existing strengths and pave the way for an inclusive and sustainable development. This new form of rural tourism in Buenos Aires province enables Villa Pardo to celebrate its cultural patrimony and beautiful natural surroundings whilst simultaneously providing for its inhabitants. It also offers its visitors a variety of authentic and low impact activities and attractions.
Enjoying local daily life and savouring local flavours
To fully appreciate Villa Pardo, it is best not be in a rush. Here, daily life happens at a slower pace than in larger towns and cities and this is part of the appeal. Visitors are encouraged to saunter around town, take in the relaxed rural atmosphere, enjoy the historic buildings and visit the local stores. Local shopkeepers will nearly always have time to share with visitors some of the many stories and anecdotes that make up the town's identity.
The promotion of local gastronomy and regional produce are key to the town's vision of rural tourism and sustainable living, and visitors will not be disappointed by the Villa Pardo many tasty offerings. These include varied menus at the hotel, regional cuisine at the local taverna and the opportunity to try traditional delicacies made by Stella Maris. Stella Maris is a local company specialising in the production of gourmet foods such as artisan cheeses and charcuterie – all perfect ingredients for picnics. There are also the recently reopened Crosta Bakery and the Lámaro grocery store to check out.
A sky full of stars in Villa Pardo, near Buenos Aires in Argentina
The town is surrounded by the fertile plains of Argentina's vast pampas and the nearby countryside is perfect for bicycle and walking excursions. The area offers great bird-watching opportunities as well as the simple pleasure of enjoying the smells and sounds of the pampas. Villa Pardo is also said to have some of the clearest skies in Buenos Aires province, making stargazing a popular activity and another way to fully appreciate the beauty and vastness of the natural world.
The home of author Adolfo Bioy Casares
It is in Villa Pardo that Adolfo Bioy Casares, author of acclaimed novel 'The Invention of Morel', spent most of his younger years and where he came to focus on his writing. The Bioy Casares family had their ranch, 'Rincon Viejo', in the nearby countryside and Adolfito, as he is known locally, enjoyed the peace and tranquility the town offered. He brought his fellow writers Jorge Luis Borges and Silvina Ocampo to Villa Pardo to spend many weekends, and these three literary greats now feature in many of the town's anecdotes.
The railway station of Villa Pardo near Buenos Aires, like travelling back in time
Today Villa Pardo's former railway station has been transformed into the Bioy Museum and Library and is the focal point for visitors to learn about the town's rich literary heritage. The old general store, built by Adolfo's grandfather, is now the 'Hotel Casa Bioy' and offers comfortable tourist accommodation with a touch of history.
Yamay, the perfect setting to reconnect to nature
On the outskirts of Villa Pardo is Yamay, the town's ecotourism centre. It is here that the concepts of rural tourism and sustainable living really come to life. In addition to being a welcoming place to stay, the property offers its guests the chance to reconnect to nature and truly appreciate simplicity. Yamay plays host to many talks and workshops on themes related to sustainable living. Guests can learn about a whole range of topics including sustainable building materials and techniques, organic farming, permaculture and recycling.
Eco-friendly accommodation in Villa Pardo, Argentina, built with natural materials and off the grid, perfect for relaxing
There is no electricity, internet, TVs or radios at the property, offering visitors a rare opportunity to disconnect from the pressures of the modern world. Accommodation is provided in two buildings constructed with adobe, a brick made from mud and other organic materials, and guests are also welcome to camp in the grounds. Yamay provides the perfect setting for guests to enjoy the beautiful surroundings and each other's company.
Visitors learning about a rural, eco-friendly lifestyle with Pardo Autentico, in Buenos Aires province
Innovative rural tourism in Argentina
Villa Pardo's innovative and successful approach to rural tourism in Buenos Aires province has received official approval and since 2015 the town has been recognised as a 'Pueblo Turistico'. This endorsement has lead to improved road access from 'Ruta Nacional No. 3' as well as new promotion opportunities.
More importantly, Villa Pardo is a positive example of how communities can work together to reverse their town's decline. Here the holistic approach has lead to job creation, a new injection of pride into the community and a revitalisation of the town's identity and traditions. Guests are able to enjoy the simple pleasures of small-town daily life – and hopefully return home with new energy and new ideas. All in all, Villa Pardo has become a rural tourism offering in Buenos Aires province that truly works for the benefit of all.
Stargazing while experiencing rural tourism in Buenos Aires province, Argentina
This post was a contribution by Hervé, a local expert from our trusted tour operator in Argentina. To learn more about the activities and adventures available in the Buenos Aires Province, you can visit our Argentina Holidays page and send a travel request.
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Vikings sign QB Sean Mannion, WR Jordan Taylor
EAGAN, Minn. (AP) — The Minnesota Vikings have signed former Los Angeles Rams quarterback Sean Mannion and ex-Denver Broncos wide receiver Jordan Taylor.
Mannion will likely replace Trevor Siemian, who joined the New York Jets, as the primary backup to Kirk Cousins. Kyle Sloter will also be in the second-string mix. Mannion was a third-round draft pick by the Rams in 2015 from Oregon State. He was the primary backup to Jared Goff last season all the way through the Super Bowl.
Undrafted out of Rice in 2015, Taylor played in 26 games for Broncos and totaled 351 yards and two touchdowns. He was sidelined last year following double hip surgery.
The Vikings also announced Monday that safety Anthony Harris and tackle Rashod Hill, both restricted free agents, signed their contract tenders.


I'm not sure if I have mentioned it before but I've always loved the Ikea Rastog utility cart. I just never had any valid reason to bring it home with me.
But thanks to the smart ladies over at Tinker Lab, I now have the perfect excuse totally practical purpose.
Woo hoo. I'm definitely getting an art cart. I mean Aggie, Aggie is definitely getting an art cart.
you don't need an excuse! I got one a while back and ended up moving every piece of furniture on my first (very small) floor to fit her in - but it was oh so worth it! I love it -so cute!
I know I shouldn't really need an excuse b/c I really do love this little cart (for reasons I can't really explain). But now I have no reason not to get it. Of course now that I've decided I'm finding all other kinds of cute similar carts elsewhere. Someone stop me!
Adorable! That could be used in a thousand different ways! These are the kinds of things we need in our homes... great multi-taskers!


Only this time there are two... The War against the Machines begins now, in Delta City!
Putting RoboCop up against Terminators sounds like such a random idea... that ic actually sounds like it can work.
Proof is, Frank Miller didn't wait a second to score both rights as he began imagining how to put both machines against one another. And he's the man behind a big part of RoboCop's cinematic franchise, so he should know, right?
Loosely based on the 1992 comic of the same name, is an action sidescroller that feels and looks like your typical run-and-gun at the time, but when you actually get down to it as a lot more in common with "bullet hells" shoot 'em up games.
The game was released on several home systems and handled consoles available back then, with the the Megadrive version being the original build of the game running on David Perry's 16-bit engine at Virgin.
RoboCop Versus The Terminator actually follows the plot of the comic to the letter (minus the drama), but is told via drastically different techniques depending on the systems it was released on.
You see, the melding between a human brain and computer chips would actually serve as the basis to propel Cyberdyne's Skynet network from simple all overseeing AI to self-aware monstrosity responsible for the war machines would wage on the entire mankind.
In the future members of the Human Resistance found another Time Machine which they used this time to destroy RoboCop. But Skynet sent several Terminators back in time as well to protect Murphy.
The game beings in the streets of Delta City facing some thugs and regular foes at first (and some Resistance soldiers I suppose) before RoboCop faces a Terminator in an alley, while after RoboCain (from the second film).
From that point RoboCop decides to go look for answers at the OCP building. He faces a new ED-209. Murphy is finally connected to Skynet, his human mind inhabiting the Skynet servers launches Judgment Day. The machines take over, allowing the war.
Now in the future, all hope is not lost. Murphy builds himself a new RoboCop body and goes after Skynet's HQ to put a stop for good... Will his cybernetic body and human soul be the different that might bring this dark future to an end?
The SNES release goes into more details regarding John Connor's soldiers sent back in time. But overall, you get to play the same game. Roughly.
RoboCop Versus The Terminator was developed by much of the same guys behind the Earthworm Jim games, which explains the similar gameplay feel, tone and even several sound effects.
The game is about 10 levels long and while it starts pretty much simple it gets rapidly overwhelming.
Enemies attack from all sides, all at the same time, continuously. Though killing them, they won't respawn so you might get some room to breath that way.
RoboCop can shot in several directions and angles, which helps when you have enemies attacking from above, but can be a bit difficult to handle when shooting near your foot.
Your enemies range from normal humans to armies of T-800 Terminators and other flying machines.
To help you out there are 8 radically different guns. The entire gameplay mechanic revolves around the two slots of available space for the weapons you can carry.
They range from RoboCop's regular automatic gun to a flame thrower, laser guns and futuristic controlled missiles. The regular gun is the less effective so the goal is to update as soon as possible. All your guns have unlimited ammo but they will be switched when you grab a new one.
But beware, when you die you will lose your current gun! So switch it back in case of low health!
The game is really, really difficult though!
Playing in easy will render your enemies simple to avoid and defeat as well as more initial lives, while playing the hardest difficulty will take out your destination's indications.
The levels are huge! And enemies will overwhelm you in numbers almost non-stop.
Each level will end with boss fights. Those are extremely difficult simple because of how many hits they require... which necessitate being careful, having patience and precision in your shots. The final boss alone against the head of Skynet (literally!) takes a gazillion hit damages to go down! It's almost as impossibly difficult as it looks ridiculous.
Some levels will have secondary objectives like helping hostages or destroying OCP cameras. That will mostly grant you more score, so no need to take those too seriously, it will only result in death.
The game looks great, and sounds great!
The music keeps pumping you up with its rapid 16-bit style beats. And the game looks dark and bloody.
The music was actually composed by the legendary Tommy Tallarico for this original version of the game.
Speaking of which it's also probably one of the goriest games I've seen on a system back then, even Splatterhouse has nothing on this!
Overall, it's a very fun, very aggressive game, but also quite frustrating.
The difficulty is really hard to manage and it gets awfully frustrating in the future levels.
The game was released on Game Gear, Master System, SNES and Game Boy at the time. A NES port was also planned but scrapped early on.
The Master System was a lot simpler albeit decent for what it aimed for.
The SNES almost looks like an entirely different game at first look. Interplay took entire control over its development. While the Sega versions seemed to borrow a lot from the films, the SNES version instead went for the look of the comic book. Featuring actual panels from its pages, and almost based entirely on the comic book art. The gameplay feels a bit worse here though as it is much slowly paced and a bit more "rigid". The graphics don't look that great (or worthy of the SNES really). The first level actually looks a lot better but after that it goes downhill fast. The spirits even appear less detailed and colored. The game was also censored (obviously, thanks Nintendo!). There are less weapons available and the default gun always takes the first slot, you can only really change the second one. RoboCain was replaced by an ED-209, but there are some new female enemies in the levels instead. Finally, its best redeeming qualities appear to be some first person shooting sequences rendered in mode 7.
All in all, this seems to be a case of a much better executed idea compared to the Dark Horse Comics original mini-series.


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La devolución de poderes es una transferencia o delegación político de una entidad a otra, o de una persona a otra. Por lo general ocurre una entidad superior a una entidad inferior se llama subsidiariedad abajo.

Por ejemplo:
 La delegación de autoridad a un gobierno soberano (véase Estructura Power);
 En el marco de la regionalización de un país (en el caso de  autonomía territorial), posiblemente con un fin federalista (que establece un Gobierno federal), la delegación a una u otra región de algunos de los poderes que previamente residía con el gobierno central.

Ejemplos
 Home Rule (Irlanda) en Irlanda.
 Devolución del poder en el Reino Unido.
 Restablecimiento del Parlamento Escocés en 1999, 300 años después de que Escocia había abandonado su parlamento nacional durante su entrada en el Reino Unido (1707).
 Establecimiento de Gobiernos autónomos en Gales y en Irlanda del Norte.
 Los Estatutos de Autonomía en España.
 Las regiones autónomas en Italia desde 2001.
 La descentralización en Francia.
 Establecimiento de la región autónoma de Nunavut en Canadá.
 Establecimiento del estatuto autonómico de Groenlandia en 2009.
 Establecimiento del régimen federal en Bélgica en 1993.

Enlaces externos

Federalismo
Descentralización

redpajama_set_name: RedPajamaWikipedia


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Iheanacho's Unsportsmanlike Conduct : Former Tottenham Midfielder, Liverpool Legend React
Former Tottenham Hotspur midfielder Tim Sherwood and ex-Liverpool winger Harry Kewell have weighed in on the controversial incident involving Kelechi Iheanacho during the first half of the ongoing game between Leicester City and Norwich City at the King Power Stadium.
On 35 minutes, the Canaries kicked the ball out of play as Buendia required treatment but Iheanacho displayed unsportsmanlike conduct when he received the throw-in as he raced towards the goal, rather than return the ball to the goalkeeper.
The Nigeria international got stamped on and was surrounded by the entire Norwich City players following the melee.
Iheanacho's conduct was a topic of discussion during the half-time analysis and Sherwood said : ''There is a challenge here on Pukki, Ricardo goes down as well, they kick the ball out.
''Ricardo picks the ball up and he tells Iheanacho to give it back to their goalkeeper. He takes it upon himself and they tackle him there, clear foul on the edge of the box.
''Obviously they feel very aggrieved for that, Norwich, I mean all over him. Todd Cantwell comes from a long way out and grabs hold of him there, scratches him, but he is going to get away with that one.
''They checked that out and in the end it was Maddison the one who kicks this one out and I think he does right, the ex-Norwich player, he had a moral ground and I think he's done the right thing''.
Kewell added : ''Iheanacho actually turns, looks and goes I am in space, give me the ball back, then he actually starts driving at the defence and you can see the reaction by the Norwich players, they are not happy but again the rule states you don't actually have to do it.''
In a tactical substitution by Leicester City manager Brendan Rodgers, Iheanacho made way for Demarai Gray in the 39th minute.


MoveStrong's special design GRT Handle is secured on the end of an Olympic Barbell with the other end inserted into a Ground Rotational Trainer, also know as a landmine. The GRT Handles rotate and allows for ease of transition from ground to overhead. It also allows hands in a neutral grip position to hold and press during the lunge and squat variation exercises as shown in this video.


Samsung Galaxy S7 is one of the most anticipated smartphones of 2016. The company's Galaxy S6 and S6 Edge flagship smartphones have been really popular this year, and so have the Galaxy Note 5 and Galaxy S6 Edge Plus phablets. The Galaxy S7 has been surfacing for months now, we've seen tons of leaks, and some of them conflict with each other, so we're not entirely sure who to believe at this point. It is quite possible, though, that the Galaxy S7 will be similar to its predecessor in terms of the overall design, and Samsung Mobile's new President said that the company is putting a heavy focus on software and services.
That being said, according to @evleaks' recent tip, Samsung actually plans to release the Galaxy S7, Galaxy S7 Edge, Galaxy S7 Edge Plus, and Galaxy S7 Plus variants of this device. Well, a new leak popped up and shared the alleged cases for the Galaxy S7 and Galaxy S7 Plus. If you take a look at the gallery down below, you'll get to see a couple of different accessories in a number of colors. Judging by the camera and flash cutout, the placement of the phone's camera will be similar to the one on the Galaxy S6, the phone's volume rockers will be placed on the left, and the power / lock button will remain on the right-hand side.
Now, this leak did not share the phone's specs, or anything of the sort, but previous leaks did. Looking at that info, the Galaxy S7 is likely to sport a 5.2-inch display once again, while the Galaxy S7 Plus might ship with a 5.7-6'' panel. Both of these handsets will sport a QHD (2560 x 1440) Super AMOLED display, and you can also expect them to ship with 4GB of RAM on the inside. Samsung's upcoming Exynos 8890 SoC will fuel these two devices, while Snapdragon 820 variants are also a possibility. These phones will be made out of metal, and the physical home button will make an appearance once again, and the fingerprint scanner will be included on the inside. Android 6.0 Marshmallow probably going to be pre-installed on this handset along with the company's TouchWiz UI.


These are the basic ingredients that make up F.G. Bradley's Sales Associates, Managers and Company Owners. If this sounds like the stuff you're made from too, we'd love to hear from you. F.G. Bradley's offers a challenging career environment, a benefits program and equal developmental opportunities for all its employees.
Resumes can be forwarded to us at jobs@fgbradleys.com or by fax to (905) 837-0624.


Our design department was asked to produce a new logo for Poplars Primary School. We decided to use a circular design featuring the poplar trees and some very happy children. The staff were extremely pleased with their re-branding and the way their request was handled.


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Dareas A. Scott / Courtesy Cydnee Patterson
Graduation Video Goes Viral for Looking Like a Movie Trailer
Cydnee Patterson found the best way to celebrate.
By Lauren Rearick
When it came time to send out the traditional graduation party invites, Cydnee Patterson of Beaumont, Texas wanted to find a unique way to reach her family and friends. Teaming up with her friend Dareas Scott of Nightfall Motion Pictures, the pair created a graduation video. And while the recent graduate, who just earned a Master of Education in Curriculum and Instruction - Reading Education from Prairie View A&M University, only intended for the virtual invitation to be a "easier, faster" option to the traditional paper method, it has since gone viral.
Her newfound internet fame occurred after Cydnee's little brother Darrell Patterson posted the video to his Twitter account, writing: "My sisters graduation video looks like a whole movie trailer." At first, her didn't tell her that he was posting the video, but then "it blew up" and he told Teen Vogue that "I just had to text my sister to let her know. She was just as shocked as me."
It's easy to see why others have enjoyed the cinematic quality of the clip. The video opens with a voice over from Cydnee explaining her struggles over the past few years, saying: "There's been times when I wanted to give up....But my faith was too big." She then recites a bible verse from Psalm 46:5, before the mood of the video quickly changes. The moving music that opens the trailer is replaced with Drake's track "God's Plan" and Cydnee is filmed walking through her university, striking poses, dancing, and smiling. The moment ends with her smashing a plastic apple filled with confetti.
According to the graduate, she chose the content of her video based on her faith. "While obtaining this degree, life brought me through a ton of ups and downs," she told Teen Vogue. "I could've let my emotions get to me and deter me away from achieving won of my goals easily. Every time I felt like I was close to that point I relied on all the things I was taught in church. I chose Drake's song "God's Plan," because everything I do, have done and will do is all apart of God's Plan. I may not always understand why I have to encounter certain experiences but I trust God is making me better."
Cydnee also explained to Teen Vogue that she has been "caught off guard" by the internet's support. "I honestly just downloaded Twitter a week prior, just so I could follow Kanye West's Tweets. And believe it or not this particular version of the video, was only meant for family to see because the introduction was so personal to me. But I'm glad to see the positive feedback that it has gotten."
Now that she's graduated with her latest degree and earned the approval of social media, Cydnee is looking forward to the future. She's planning to apply for a Doctorate program, and also has some advice for others pursuing their educational endeavors. "Don't give up, no matter how bad it may get the reward at the end is better," she said. "You may have to miss out on a few social events here or there but that's a temporary sacrifice for your future self. Stay the course and have faith and a little patience. Celebrate every one of your accomplishments whether big or small. Trust God."
Related: Viral Graduation Video Shows Dad Interviewing His Daughter Every First Day of School
Keywordsgraduation
North West's Two-Tone Hair Is Too Cute
She's definitely the cutest influencer bringing this trend into 2023.
"Ginny & Georgia" Giving Us More Bracia Is Season 2's Best Change
Season 2 Bracia proves she's more than Ginny's token Black friend.
By Liv Facey
A M3GAN Sequel Is Coming in 2025
"M3GAN 2.0" will be back for blood.
Before She Was M3GAN, Jenna Davis Was the "Penny Nickel Dime" Girl
"I guess publicity is publicity in a way."


First of all, i make this because sometimes there are new players asking where is the "gym" and i only tell them to go look for any map with "city" on the name, but i haven't even asked them if they know how to use the maps on this game, and now that we're clear on that, here comes the bulk of this thread.
You can still go to the Search Maps page if you click on the "click here to see this zone detailed" button(with the additional benefit of sorting the maps by the zone you selected, i'll explain that later), but don click on the "click here yo see this zone's full size image" one, it just directs you to deviantart and it will only waste your time.
Filter By Zone: this will let you look at the all the maps of a certain zone and only of that zone, if the map you are looking for isn't in that zone, you will not find it, this may be useful for players that are looking for the arena of any zone.
Filter By Route Name: this is only useful if you know the name of the map you are looking for, but ignore where it is or how to reach it, useful for those "last level gained on X" transformations.
Filter By AVG Level Min/Max: this lets you look for maps with the average level of encounters, Min for the minimum and above, and Max for the maximum and below, if you input a number on both, it will show you the maps with encounters between the numbers you input(all of this excluding Zenith, Legendary and Ancient class encounters), useful for looking for maps that don't exceed any level and not worry about battling overwhelming enemies(rare classes notwithstanding).
Filter By Monster Name/Id: this will let you look for all maps with the encounters you are looking for, be it by name or by id number, useful for that exact purpose.
Contains Zenith: it lets you choose if you want maps with this class on them or not, All shows all maps regardless of it containing the class, Yes shows only maps with them and No shows maps withouth them.
Contains Legendary: same as above, but for legendary class.
Contains Ancient: same as above, keep in mind you can combine them to find maps with only Ancient or maps with Zenith and Legendary, or even maps with none of them.
Sort By Route Name: this sort the maps by alphabetical order, Ascending for normal order and Descending for reversed order, if you haven't choosen any, the maps are ordered by the route number.
Sort By Route Order: same as above, but this time the order is numerical.
The sorting is by alphabetical order and it shows maps with only Ancient class in them(other than normal encounters of course), by the way, the image shows only a part of the maps that appear.
Other than all that, i can only tell you to look for arenas in maps with "city" on their name and look for guardians in the last map of each zone(except the last one, that one's in the second to last map), i hope you find this useful, and if you didn't understand something or i forgot some details i might be adding here(may as well shrink the images a bit), be sure to tell me, be it here or in-game chat(same username).
no thanks, i didnt do this to get rewards, but i want to ask, is there a way to translate other guides or start them anew for spanish readers and post them in this forum?


New, size 6.5m, over $100 value, asking $75 obo. call angela.
Size 9-1/2, peanut brittle lizard. like new!
With yellow accents, still in the box.
Black, size 12. with a mesh tote bag.
Name brand, are very clean and in good condition.
1 size 8, 2 are size 9, 1 size 10.5, 1 size 9.5, 1 size 12.
Pretty blush color, velcro closure. like new!


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Höfði Reykjavík Peace Centre receives a grant to develop intensive courses in peace and conflict studies
Höfði Reykjavík Peace Centre at the University of Iceland has received a grant from the Nordplus Higher Education programme to develop intensive courses in peace and conflict studies. The first one-week intensive course will take place in Tromsö, Norway, in spring 2020, the second one in Tampere, Finland, fall 2020, and the third and final one will take place in Iceland spring 2021. The courses mark the first step in the development of a masters programme in peace and conflict studies at the University of Iceland.
The project, People, Communities and Cities in Peacebuilding: An Inclusive and Intersectional Approach to Peace Studies (InPeace), designs innovative intensive courses in peace studies, building on unconventional and critical approaches to peace processes. It draws on femininities, masculinities and intersectionality, allowing for improved understanding of conflicts in the current state of affairs.
Sub- and non-state actors are increasingly relevant internationally, exemplified by sanctuary cities in the US, social movements like #metoo, various insurgencies in Afghanistan, IS in Iraq/Syria, or maoist movements in India. This phenomenon emerges simultaneously with increased nationalism and extremism, where populist parties and politicians have gained in global prominence. Many of these actors represent "grassroot" movements, reflecting the political interests of people who have otherwise not felt represented by the state. In response, renewed approaches in peace and conflict studies are needed, focusing on peacebuilding "from below", acknowledging gender and other categories, understanding "the local" in peace processes, and sub/non-state actor roles as either spoilers or supporters of peace.
The InPeace project creates an innovative network of university-based peace institutes in the Nordic region, strengthening the position of Nordic universities in peace research and education. InPeace develops new teaching material through three intensive courses in Tromsø, Tampere, and Reykjavík.
The project is coordinated by Höfði Reykjavík Peace Centre, in collaboration with Tampere Peace Research Institute (TAPRI) at the University of Tampere and the Centre for Peace Studies (CPS) at UiT – The Arctic University of Norway.
Pia Hansson, Director of Höfði Reykjavik Peace Centre, Auður Örlygsdóttir, Project Manager at Höfði Reykjavík Peace Centre, Silja Bára Ómarsdóttir, Associate Professor in Political Science, and Guðrún Sif Friðriksdóttir, PhD student in Anthropology at the University of Iceland, will coordinate it on behalf of Höfði Reykjavík Peace Centre and the University of Iceland, in cooperation with professors from the partner universities.


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T.I.'s Paper Trail Loses Billboard #1 To Country Star Kenny Chesney's Latest
New LPs from Ray LaMontagne, Keane and Lucinda Williams debut in next week's top 10.
archive-Chris-Harris 10/22/2008
Justin Timberlake Looks Back To The Future; Plus Pitbull, Common, Keane And More, In New Releases
Also due Tuesday: Chuck D, Mudvayne, Incubus, T.H. White, Simply Red, As Tall as Lions, Flogging Molly.
archive-Kurt-Orzeck 11/26/2007
For The Record: Quick News On Hilary Duff, Busta Rhymes, Fat Joe, The Game, Cassie, Diddy, Keane & More
Hilary heading to New Orleans; Busta still won't talk about bodyguard murder; basketball pits Game against Fat Joe.
New Releases: Nelly Furtado, Madonna, Korn, Keane, Underoath, Three 6 Mafia, Pearl Jam & More
Also due Tuesday: Motion City Soundtrack, Frank Black, Stuart Staples, Counting Crows, Field Mob, Luna.
Keane Nearly Followed Breakthrough With A Breakup
Piano rockers persevered, however, to finish second LP, Under the Iron Sea.
James Montgomery 04/25/2006
Why (Fill In The Blank) Deserves The Best New Artist Grammy
Ciara, Fall Out Boy, Keane, John Legend, Sugarland to lock horns for award made famous by ... er, Milli Vanilli?
archive-Corey-Moss 01/31/2006
Keane Get Over Their Fears, Want The Funk Instead
English trio trying to avoid treading familiar ground on second LP.
Gil Kaufman 06/15/2005
Franz Ferdinand, Scissor Sisters, Keane Win Big At Brit Awards
Sisters, an American group, lead the pack with three awards.
Keane Gearing Up To Record New LP, Tour Their Fingers Off
Piano rockers hitting the studio in April, hitting the road most of the year.
archive-Alyssa-Rashbaum 02/04/2005
Just Because A Piano Can't Move Doesn't Mean Keane Don't
Brits have been known to rock so hard onstage they need an oxygen tank.
With Coldplay And Darkness On Board, Band Aid Is Revived For Charity Single
Proceeds will go toward famine relief in Ethiopia and the Sudan.