Pairwise ratio-based differential abundance analysis of infant microbiome 16S sequencing data.

Differential abundance analysis of infant 16S microbial sequencing data is complicated by challenging data properties, including high sparsity, extreme dispersion and the relative nature of the information contained within the data. In this study, we propose a pairwise ratio analysis that uses the compositional data analysis principle of subcompositional coherence and merges it with a beta-binomial regression model. The resulting method provides a flexible and easily interpretable approach to infant 16S sequencing data differential abundance analysis that does not require zero imputation. We evaluate the proposed method using infant 16S data from clinical trials and demonstrate that the proposed method has the power to detect differences, and demonstrate how its results can be used to gain insights. We further evaluate the method using data-inspired simulations and compare its power against related methods. Our results indicate that power is high for pairwise differential abundance analysis of taxon pairs that have a large abundance. In contrast, results for sparse taxon pairs show a decrease in power and substantial variability in method performance. While our method shows promising performance on well-measured subcompositions, we advise strong filtering steps in order to avoid excessive numbers of underpowered comparisons in practical applications.

Longitudinal study on the prevalence of extended spectrum cephalosporins-resistant Escherichia coli colonization in Dutch veal farms.

A longitudinal study was performed to investigate the prevalence of Extended-Spectrum Cephalosporin-Resistant (ESC-R) Escherichia coli colonization in Dutch veal farms. Rectal swabs from 683 calves born in 13 Dutch dairy farms were collected one day prior to transportation to the veal farm at 14 or 28 days of age, and at 5 different time points 8 Dutch veal farms. In addition, characteristics of the calf, cows, and farm management were collected. Rectal swabs were selectively cultured for ESC-R E. coli. In total, 1202 ESC-R E. coli isolates were recovered. Overall, the prevalence of ESC-R E. coli increased from 24.4 % at one day prior to transportation to 57.3 % in week two after arrival of calves at the veal farm. No associations were found between the presence of ESC-R E. coli at the dairy or veal farm and age of transportation, sex and breed. The presence of ESC-R E. coli in week 6, 10, and 18 at the veal farm was positively associated with the presence of ESC-R E. coli in week 10, 18, and 24, respectively (p < 0.05). Individual antibiotic treatments applied before week 2 and 6 upon arrival to the veal farms tended to increase the ESC-R E. coli colonization frequency. Our results indicate that ESC-R E. coli colonization frequency substantially increases after arrival of calves on the veal farm. In addition to individual antibiotic treatments, it is considered likely that frequently applied batch antibiotic treatments are also implicated in the ESC-R E. coli colonization frequency.

Developing a feasible and sensitive judgement bias task in dairy cows.

Judgement bias tasks (JBTs) are used to assess the influence of farm practices on livestock affective states. The tasks must be adjusted to the species and age group of focus. In cattle, most JBTs were designed for calves instead of adult cows. This study aimed to develop a JBT suitable for adult dairy cows, combining feasibility, validity, sensitivity and repeatability. Three JBTs were developed in which cows were trained to reach or avoid reaching a feeder, the location of which signalled a reward or punisher. The tasks differed in terms of punisher-cows being allocated either to "no-reward", an air puff or an electric shock. Cows were then exposed twice to three ambiguous positions of the feeder, on two separate occasions. Speed of learning and proportions of correct responses to the conditioned locations were used to assess the feasibility of the task. Adjusted latencies to reach the ambiguous feeder positions were used to examine whether response patterns matched the linear and monotonic graded pattern expected in a valid and sensitive JBT at baseline. Latencies to reach the feeders in the two repeated testing sessions were compared to assess ambiguity loss over tasks' repetitions. The validity of using spatial JBTs for dairy cows was demonstrated. While the effect on JBT feasibility was nuanced, the punisher did influence JBT sensitivity. None of the JBTs' repeatability could be supported. We conclude that using an air puf as punisher led to the most sensitive, yet non-repeatable, JBT for dairy cows.

Housing and personality effects on judgement and attention biases in dairy cows.

Affective states can be inferred from responses to ambiguous and threatening stimuli, using Judgement Bias Tasks (JBTs) and Attention Bias Tasks (ABTs). We investigated the separate and interactive effects of personality and housing conditions on dairy cattle affective states. We assessed personality in 48 heifers using Open-Field, Novel-Object and Runway tests. Personality effects on responses to the JBT and to the ABT were examined when heifers were housed under reference conditions. Heifers were subsequently housed under positive or negative conditions, and housing effects on animal responses in both tasks were investigated while controlling for personality. A Principal Component Analysis revealed three personality traits labelled Activity, Fearfulness and Sociability. Under reference conditions, personality influenced heifers' responses to the JBT and to the ABT, therefore questioning the tasks' generalizability across individuals. Against expectations, housing did not influence responses to the  JBT and heifers in the negative conditions looked at the threat later than heifers in the positive or reference conditions. More research is warranted to confirm the validity and the repeatability of the JBT and of the ABT as appropriate measures of affective states in dairy cows.

Effects of pre-transport diet, transport duration and transport condition on immune cell subsets, haptoglobin, cortisol and bilirubin in young veal calves.

The aim of this study was to investigate effects of pre-transport diets, transport durations and transport conditions on immune cell subsets, haptoglobin, cortisol and bilirubin of young calves upon arrival at the veal farm. An experiment was conducted with a 2 × 2 × 2 factorial arrangement with 3 factors: 1) provision of rearing milk or electrolytes at the collection center (CC); 2) transport duration (6 or 18 hours) and 3) transport condition (open truck or conditioned truck). Holstein-Friesian and cross-bred calves were used (N = 368; 18 ± 4 days; 45.3 ± 3.3 kg). Blood samples were collected from calves (N = 128) at the collection center, immediately post-transport (T0) and 4, 24, 48 hours, week 1, 3 and 5 post-transport. Blood was analyzed for cortisol, bilirubin, haptoglobin, IgG and IgM. Moreover, cell counts of neutrophils, lymphocytes, monocytes, basophils and eosinophils were measured in blood samples taken at the collection center and T0. In these same blood samples, different lymphocyte populations were characterized by flow cytometry, including CD14+ cells, NK cells, δγ+ T cells, CD8+ cells, CD4+ cells and CD21+ cells. Calves transported in the conditioned truck had higher amounts of white blood cell count (WBC) (Δ = 1.39 × 109/l; P = 0.01), monocytes (Δ = 0.21 × 109/l; P = 0.04), neutrophils (Δ = 0.93 × 109/l; P = 0.003), than calves transported in the open truck regardless, of pre-transport diet or transport duration. The study showed that transport condition and duration influenced parts of the innate immune system of young veal calves. Cortisol, bilirubin and WBC seemed to be connected by similar underlying mechanisms in relation to transport conditions. However, it is unclear which specific pathways in the immune system of young calves are affected by different transport conditions (e.g. temperature, humidity, draught).

Human spatial memory implicitly prioritizes high-calorie foods.

All species face the important adaptive problem of efficiently locating high-quality nutritional resources. We explored whether human spatial cognition is enhanced for high-calorie foods, in a large multisensory experiment that covertly tested the location memory of people who navigated a maze-like food setting. We found that individuals incidentally learned and more accurately recalled locations of high-calorie foods - regardless of explicit hedonic valuations or personal familiarity with foods. In addition, the high-calorie bias in human spatial memory already became evident within a limited sensory environment, where solely odor information was available. These results suggest that human minds continue to house a cognitive system optimized for energy-efficient foraging within erratic food habitats of the past, and highlight the often underestimated capabilities of the human olfactory sense.

Transport of Young Veal Calves: Effects of Pre-transport Diet, Transport Duration and Type of Vehicle on Health, Behavior, Use of Medicines, and Slaughter Characteristics.

The aim of this study was to investigate effects of different early life transport-related factors on health, behavior, use of medicines and slaughter characteristics of veal calves. An experiment was conducted with a 2 × 2 × 2 factorial arrangement with 3 factors: (1) provision of rearing milk or electrolytes before transport, (2) transport duration (6 or 18 h), and (3) type of vehicle (open truck or conditioned truck). The study included male Holstein-Friesian and cross-bred calves (N = 368; 18 ± 4 days; 45.3 ± 3.3 kg). Data on health status of calves were collected at the collection center and at the veal farm until week 27 post-transport. Behavior of calves was recorded during transport and at the veal farm until week 13 post-transport. Use of herd and individual medical treatments was recorded at the veal farm. The prevalence of loose or liquid manure at the veal farm from day 1 until week 3 post-transport was lower in electrolyte-fed calves transported in the conditioned truck compared to electrolytes-fed calves transported in the open truck or milk-fed calves transported in both the conditioned and open truck (Δ = 11% on average; P = 0.02). In comparison with the open truck, calves transported in the conditioned truck had lower prevalence of navel inflammation in the first 3 weeks post-transport (Δ = 3 %; P = 0.05). More milk-fed calves received individual antibiotic treatments compared to electrolyte-fed calves at the veal farm (P = 0.05). In conclusion, the transport-related factors examined in the present study affected health and behavior of calves in the short-term, but there was no evidence for long-term effects. It remains unknown why no long-term effects were found in this study. Perhaps this absence of transport-related effects was due to multiple use of medical treatments in the first weeks at the veal farm. Alternatively, it might be that the collective effects of the transition from the dairy farm to the veal farm, and of the husbandry conditions during the subsequent rearing period, on the adaptive capacity of calves were so large that effects of individual transport-related factors were overruled.

The effect of intensive grazing systems on the rising plate meter calibration for perennial ryegrass pastures.

The rising plate meter (RPM) is used to measure grass height, which subsequently is used in a calibration equation to estimate herbage mass (HM), an important parameter for optimization of feed management in grazing systems. The RPM is placed on the sward and measures the resistance of the sward toward the plate, which depends not only on grass length, but also on sward structure. The accuracy of the calibration equation for the RPM to estimate HM across grazing systems, however, has not yet been evaluated. Therefore, our aim was to analyze the effect of intensive grazing systems on RPM calibration for perennial ryegrass pastures. To do so, we studied 2 grazing systems: compartmented continuous grazing (CCG) and strip grazing (SG), which differ in key grazing characteristics, such as pre- and post-grazing heights and period of regrowth, that may influence tiller density and vertical flexibility of the sward. The experiment was performed from April until October in 2016 and 2017 with 60 dairy cows, at a fixed stocking rate of 7.5 cows per hectare. To calibrate the RPM, 256 direct measurements of HM >4 cm (i.e., above stubble) were collected by cutting and weighing plots of grass for CCG and SG. Our main interest was in the HM above stubble because this is consumed by cows. Herbage mass <4 cm represents the stubble left after grazing. Differences in HM <4 cm may (partially) explain differences in HM >4 cm between the grazing systems. Therefore, HM <4 cm was additionally measured on 4 out of every 8 plots per grazing system by cutting out quadrats to 0 cm with an electric grass trimmer. Our results showed an average error margin in our calibration equations of 25 to 31%, expressed as the root mean square error of prediction (RMSEP) as a percentage of the observed HM >4 cm. Differences between grazing systems were relatively small, and including grazing system as a factor in the regression model to explain the increase in HM per centimeter of grass did not reduce the RMSEP of the model to any relevant extent. On the other hand, HM <4 cm was significantly greater on CCG compared with SG, with 2,042 kg of DM per hectare for CCG and 1,676 kg of DM per hectare for SG. The HM <4 cm, however, is not used for grazing, and this difference was not reflected in the HM >4 cm. Our results indicate that we can use one region-specific calibration equation for perennial ryegrass pastures across intensive grazing systems, despite relatively large differences in pre- and post-grazing heights and period of regrowth.

Correcting fresh grass allowance for rejected patches due to excreta in intensive grazing systems for dairy cows.

Dairy farms with intensive grazing systems combine grazing with supplemental feeding, which can be challenging because an incorrect balance between fresh grass allowance and feed supplementation results in inefficient use of the pasture, lower feed efficiency, and potential decreases in animal production. When estimating fresh grass allowance, we currently do not correct for the formation of rejected patches (RP) surrounding excreta, which can lead to overestimation of the potential fresh grass intake and hampers optimal grazing. In this study, therefore, we aim to quantify the formation of RP in intensive grazing systems and improve the quantification of fresh grass allowance. To do so, we studied 2 grazing systems (i.e., compartmented continuous grazing and strip grazing) that differ in key grazing characteristics, such as pre- and post-grazing heights and period of regrowth. The experiment was performed from April to October in 2016 and 2017 with 60 dairy cows at a fixed stocking rate of 7.5 cows/ha. Average pre-grazing grass height was measured with a rising plate meter. To quantify the formation of RP after grazing, individual grass height measurements were conducted after grazing and classified as RP or not, based on visual assessment. Our analysis showed that the average percentage of grassland covered with RP increased from around 22% at the end of May to around 43% at the end of July/beginning of August, and these percentages do not differ across grazing systems. The percentage of grassland covered with RP should be subtracted from the total grazed area to better estimate true fresh grass allowance.

Study of potential health effects of electromagnetic fields of telephony and Wi-Fi, using chicken embryo development as animal model.

The objective of this study is to investigate possible biological effects of radiofrequency electromagnetic fields (RF-EMF) as used in modern wireless telecommunication in a well-controlled experimental environment using chicken embryo development as animal model. Chicken eggs were incubated under continuous experimental exposure to GSM (1.8 GHz), DECT (1.88 GHz), UMTS (2.1 GHz), and WLAN (5.6 GHz) radiation, with the appropriate modulation protocol, using a homogeneous field distribution at a field strength of approximately 3 V/m, representing the maximum field level in a normal living environment. Radiation-shielded exposure units/egg incubators were operating in parallel for exposed and control eggs in a climatized homogeneous environment, using 450 eggs per treatment in three successive rounds per treatment. Dosimetry of the exposure (field characteristics and specific absorption rate) were studied. Biological parameters studied included embryo death during incubation, hatching percentage, and various morphological and histological parameters of embryos and chicks and their organs, and gene expression profiles of embryos on day 7 and day 18 of incubation by microarray and qPCR. No conclusive evidence was found for induced embryonic mortality or malformations by exposure to the used EMFs, or for effects on the other measured parameters. Estimated differences between treatment groups were always small and the effect of treatment was not significant. In a statistical model that ignored possible interaction between rounds and exposure units, some of the many pairwise comparisons of exposed versus control had P-values lower than 0.05, but were not significant after correction for multiple testing. Bioelectromagnetics. 38:186-203, 2017. © 2017 Wiley Periodicals, Inc.

Feeding guild of non-host community members affects host-foraging efficiency of a parasitic wasp.

Interactions between predator and prey, or parasitoid and host, are shaped by trait- and density-mediated processes involving other community members. Parasitoids that lay their eggs in herbivorous insects locate their hosts through infochemicals such as herbivore-induced plant volatiles (HIPVs) and host-produced kairomones. Hosts are frequently accompanied by non-host herbivores that are unsuitable for the parasitoid. These non-hosts may interfere with host location primarily through trait-mediated processes, by their own infochemicals, and their induction of the emission of plant volatiles. Although it is known that single non-hosts can interfere with parasitoid host location, it is still unknown whether the observed effects are due to species specific characteristics or to the feeding habits of the non-host herbivores. Here we addressed whether the feeding guild of non-host herbivores differentially affects foraging of the parasitoid Cotesia glomerata for its common host, caterpillars of Pieris brassicae feeding on Brassica oleracea plants. We used different phloem-feeding and leaf-chewing non-hosts to study their effects on host location by the parasitoid when searching for host-infested plants based on HIPVs and when searching for hosts on the plant using infochemicals. To evaluate the ultimate effect of these two phases in host location, we studied parasitism efficiency of parasitoids in small plant communities under field-tent conditions. We show that leaf-chewing non-hosts primarily affected host location through trait-mediated effects via plant volatiles, whereas phloem-feeding non-hosts exerted trait-mediated effects by affecting foraging efficiency of the parasitoid on the plant. These trait-mediated effects resulted in associational susceptibility of hosts in environments with phloem feeders and associational resistance in environments with non-host leaf chewers.

Marker-based estimation of heritability in immortal populations.

Heritability is a central parameter in quantitative genetics, from both an evolutionary and a breeding perspective. For plant traits heritability is traditionally estimated by comparing within- and between-genotype variability. This approach estimates broad-sense heritability and does not account for different genetic relatedness. With the availability of high-density markers there is growing interest in marker-based estimates of narrow-sense heritability, using mixed models in which genetic relatedness is estimated from genetic markers. Such estimates have received much attention in human genetics but are rarely reported for plant traits. A major obstacle is that current methodology and software assume a single phenotypic value per genotype, hence requiring genotypic means. An alternative that we propose here is to use mixed models at the individual plant or plot level. Using statistical arguments, simulations, and real data we investigate the feasibility of both approaches and how these affect genomic prediction with the best linear unbiased predictor and genome-wide association studies. Heritability estimates obtained from genotypic means had very large standard errors and were sometimes biologically unrealistic. Mixed models at the individual plant or plot level produced more realistic estimates, and for simulated traits standard errors were up to 13 times smaller. Genomic prediction was also improved by using these mixed models, with up to a 49% increase in accuracy. For genome-wide association studies on simulated traits, the use of individual plant data gave almost no increase in power. The new methodology is applicable to any complex trait where multiple replicates of individual genotypes can be scored. This includes important agronomic crops, as well as bacteria and fungi.

Chopped or long roughage: what do calves prefer? Using cross point analysis of double demand functions.

The present study aimed to quantify calves' (Bos taurus) preference for long versus chopped hay and straw, and hay versus straw, using cross point analysis of double demand functions, in a context where energy intake was not a limiting factor. Nine calves, fed milk replacer and concentrate, were trained to work for roughage rewards from two simultaneously available panels. The cost (number of muzzle presses) required on the panels varied in each session (left panel/right panel): 7/35, 14/28, 21/21, 28/14, 35/7. Demand functions were estimated from the proportion of rewards achieved on one panel relative to the total number of rewards achieved in one session. Cross points (cp) were calculated as the cost at which an equal number of rewards was achieved from both panels. The deviation of the cp from the midpoint (here 21) indicates the strength of the preference. Calves showed a preference for long versus chopped hay (cp = 14.5; P = 0.004), and for hay versus straw (cp = 38.9; P = 0.004), both of which improve rumen function. Long hay may stimulate chewing more than chopped hay, and the preference for hay versus straw could be related to hedonic characteristics. No preference was found for chopped versus long straw (cp = 20.8; P = 0.910). These results could be used to improve the welfare of calves in production systems; for example, in systems where calves are fed hay along with high energy concentrate, providing long hay instead of chopped could promote roughage intake, rumen development, and rumination.

Assessment of performance of selected serological tests for diagnosing brucellosis in pigs.

Swine brucellosis due to Brucella suis is considered an emerging zoonotic disease whose control is based on serological testing and the subsequent culling of seropositive animals or the full depopulation of affected flocks. Here we assessed the performance of several serological tests (Rose Bengal Test [RBT], indirect ELISA [i-ELISA], blocking ELISA [b-ELISA], and two competitive ELISAs [c-ELISA]) for diagnosing swine brucellosis caused by B. suis biovar 2. Both frequentistic and Bayesian statistical inference were used. A frequentistic analysis, using sera from known gold standard (GS) populations (i.e., from truly infected or brucellosis free animals), resulted in maximum (100%) diagnostic sensitivity (Se) and specificity (Sp) in the RBT, i-ELISA and b-ELISA tests. However, c-ELISAs resulted in lower diagnostic Se (ranging from 68.5% to 92.6%, according to the different cut-offs selected). A Bayesian analysis of tests yielding the best diagnostic performance with GS sera (RBT, i-ELISA and b-ELISA), but using a large collection of field sera, resulted in similar Se among tests but markedly lower (≈ 80%) than that resulting from the frequentistic analysis using the GS serum populations. By contrast, the estimated Sp in the Bayesian analysis was only slightly lower than 100%, thus similar to that obtained frequentistically. Our results show that adequate diagnostic tests for brucellosis in swine are available, but also emphasize the need for more extensive validation studies before applying these tests under field conditions.

Actinobacillus pleuropneumoniae is impaired by the garlic volatile allyl methyl sulfide (AMS) in vitro and in-feed garlic alleviates pleuropneumonia in a pig model.

Decomposition products of ingested garlic are to a certain extent excreted via the lungs. If the supposed health-supporting capacities associated with garlic extend to these exhaled sulfurous compounds, they could have an effect on the course of pneumonia. In this study, the garlic-derived volatile allyl methyl sulfide (AMS) as a lead compound of volatile garlic metabolites was shown to exhibit an antibacterial effect against the pig pathogen Actinobacillus pleuropneumoniae serotype 9. AMS caused a delay in the appearance of the optical density-monitored growth of A. pleuropneumoniae in medium when compared to unaffected growth curves, yet without lowering the stationary phase yield at the concentration range tested. At 1.1mM, AMS impaired the in vitro growth rate of A. pleuropneumoniae serotype 9 by 8% compared to unimpeded growth. In an animal trial, a garlic-fed group of 15 pigs that received a diet with 5% garlic feed component and a control group of 15 pigs that received a diet without garlic were infected with A. pleuropneumoniae serotype 2 via an aerosol and subsequently followed for 4 days. At the day of the challenge, blood AMS in the garlic-fed group amounted to 0.32 ± 0.13 µM. A beneficial, alleviating effect of garlic on the course and severity of an A. pleuropneumoniae infection in pigs was indicated by the reduced occurrence of characteristic pleuropneumonia lesions (27% of the lungs affected in the garlic-fed group vs. 47% in the control group) and a near to significant (p=0.06) lower relative lung weight post mortem in the garlic-fed group.

Breeding with resistant rams leads to rapid control of classical scrapie in affected sheep flocks.

Susceptibility to scrapie, a transmissible spongiform encephalopathy in sheep, is modulated by the genetic make-up of the sheep. Scrapie control policies, based on selecting animals of resistant genotype for breeding, have recently been adopted by the Netherlands and other European countries. Here we assess the effectiveness of a breeding programme based on selecting rams of resistant genotype to obtain outbreak control in classical scrapie-affected sheep flocks under field conditions. In six commercially-run flocks following this breeding strategy, we used genotyping to monitor the genotype distribution, and tonsil biopsies and post-mortem analyses to monitor the occurrence of scrapie infection. The farmers were not informed about the monitoring results until the end of the study period of six years. We used a mathematical model of scrapie transmission to analyze the monitoring data and found that where the breeding scheme was consistently applied, outbreak control was obtained after at most four years. Our results also show that classical scrapie control can be obtained before the frequency of non-resistant animals is reduced to zero in the flock. This suggests that control at the national scale can be obtained without a loss of genetic polymorphisms from any of the sheep breeds.

Postnatal development of collagen structure in ovine articular cartilage.

BACKGROUND: Articular cartilage (AC) is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Across species, adult AC shows an arcade-like structure with collagen predominantly perpendicular to the subchondral bone near the bone, and collagen predominantly parallel to the articular surface near the articular surface. Recent studies into collagen fibre orientation in stillborn and juvenile animals showed that this structure is absent at birth. Since the collagen structure is an important factor for AC mechanics, the absence of the adult Benninghoff structure has implications for perinatal AC mechanobiology. The current objective is to quantify the dynamics of collagen network development in a model animal from birth to maturity. We further aim to show the presence or absence of zonal differentiation at birth, and to assess differences in collagen network development between different anatomical sites of a single joint surface. We use quantitative polarised light microscopy to investigate properties of the collagen network and we use the sheep (Ovis aries) as our model animal. RESULTS: Predominant collagen orientation is parallel to the articular surface throughout the tissue depth for perinatal cartilage. This remodels to the Benninghoff structure before the sheep reach sexual maturity. Remodelling of predominant collagen orientation starts at a depth just below the future transitional zone. Tissue retardance shows a minimum near the articular surface at all ages, which indicates the presence of zonal differentiation at all ages. The absolute position of this minimum does change between birth and maturity. Between different anatomical sites, we find differences in the dynamics of collagen remodelling, but no differences in adult collagen structure. CONCLUSIONS: The collagen network in articular cartilage remodels between birth and sexual maturity from a network with predominant orientation parallel to the articular surface to a Benninghoff network. The retardance minimum near, but not at, the articular surface at all ages shows that a zonal differentiation is already present in the perinatal animals. In these animals, the zonal differentiation can not be correlated to the collagen network orientation. We find no difference in adult collagen structure in the nearly congruent metacarpophalangeal joint, but we do find differences in the dynamics of collagen network remodelling.

Scrapie prevalence in sheep of susceptible genotype is declining in a population subject to breeding for resistance.

BACKGROUND: Susceptibility of sheep to scrapie infection is known to be modulated by the PrP genotype of the animal. In the Netherlands an ambitious scrapie control programme was started in 1998, based on genetic selection of animals for breeding. From 2002 onwards EU regulations required intensive active scrapie surveillance as well as certain control measures in affected flocks.Here we analyze the data on genotype frequencies and scrapie prevalence in the Dutch sheep population obtained from both surveillance and affected flocks, to identify temporal trends. We also estimate the genotype-specific relative risks to become a detected scrapie case. RESULTS: We find that the breeding programme has produced a steady increase in the level of genetic scrapie resistance in the Dutch sheep population. We also find that a significant decline in the prevalence of scrapie in tested animals has occurred a number of years after the start of the breeding programme. Most importantly, the estimated scrapie prevalence level per head of susceptible genotype is also declining significantly, indicating that selective breeding causes a population effect. CONCLUSIONS: The Dutch scrapie control programme has produced a steady rise in genetic resistance levels in recent years. A recent decline in the scrapie prevalence per tested sheep of susceptible prion protein genotype indicates that selective breeding causes the desired population effect.

Serodiagnosis of Mycobacterium avium infections in pigs.

The aim of this study is the development and evaluation of a serodiagnostic assay for Mycobacterium avium (MA). After screening MA lipid fractions in an ELISA format, a polar lipid fraction was selected as antigen because of its superior recognition by serum antibodies in experimentally infected pigs. The resulting MA-ELISA was evaluated as an alternative for detection of MA infection by traditional pathological examination of pig lymph nodes for granulomatous lesions by meat inspectors. By comparing with bacteriological examination, the MA-ELISA showed significantly better sensitivity (69%) as compared to pathological examination (31%) in experimentally infected pigs. The MA-ELISA also appeared significantly more specific in a set of serum samples from MA negative pigs: only 1 out of these 153 serum samples reacted positive, whereas 99 (65%) of these had displayed false positive results by detection of lymph nodes lesions that appeared not to be associated with MA (Komijn et al., 2007). The MA-ELISA was subsequently evaluated using serum samples from two farms with pigs known to be infected with MA. Bacteriological examination of the sub-maxillary and mesenteric lymph nodes showed that 56% (103/184) and 35% (41/117) of the pigs, respectively were positive for MA in these farms. In the first farm, 16% (29/184) of the pigs tested positive in MA-ELISA and 31% (57/184) by pathological examination. On the contrary, in the second farm, more pigs tested positive 17% (15/117) in MA-ELISA with 8% (9/117) positivity by pathological examination. Taking the results on both farms together, the sensitivity of the MA-ELISA was 14% and the specificity 83%, whereas the sensitivity of the pathological examination was 31% and the specificity 86%. For practical reasons use of a serological test as the MA-ELISA may be preferred over pathological or bacteriological examinations. Our studies in experimentally infected and negative "field" sera indicate that the MA-ELISA is significantly more specific and more sensitive than detection by classical pathological examination. However, the studies in two MA infected farms show a variable picture with pathological examination overall performing better. Study in a wider range of "positive" farms will be needed to provide a more comprehensive view of the quality of both tests for detection of MA in infected farms. At the same time further optimization of MA-ELISA with use of lipid antigens from a broader range of serotypes may improve its performance in the face of infections with different MA serotypes.