Implementing a genomic rotational crossbreeding scheme to promote local dairy cattle breeds-A simulation study.

In dairy cattle breeding, there is a clear trend toward the use of only a few high-yielding breeds. One main reason is that efficient breeding programs require a certain population size. Since some numerically small breeds are well known for their functional traits, they might be an interesting crossing partner for high-yielding breeds with the aim to utilize heterosis. This simulation study investigated the transition period of a small cattle population for the implementation of genomic selection and rotational crossbreeding with a high-yielding breed. Real SNP chip genotype data from the numerically small red dairy breed Angler and the high-yielding breed Holstein Friesian were used to simulate the base generations, from which rotational crossbreeding was conducted for 10 generations. A polygenic trait with many quantitative trait loci with additive and directional dominance effects was simulated. Different selection methods for Angler sires (purebred performance, crossbred performance, and weighted purebred-crossbred performance) and different sizes and structures of the reference population (Angler, crossbred animals, and Angler plus crossbred animals) were considered. The results showed that the implementation of a genomic rotational crossbreeding scheme is an appealing option to promote the numerically small Angler breed. The growing reference population consisting of Angler and crossbred individuals maximized the genetic gain for Angler and the performance level for the crossbred individuals. Selection for purebred performance, crossbred performance, or a weighted combination of both hardly affected the results, and differences between selection scenarios were observed only in the long term with decreasing purebred-crossbred correlations.

Derivation of economic values for German dairy breeds by means of a bio-economic model-with special emphasis on functional traits.

To assess the economic importance of breeding traits, economic values (EV) were derived for 3 German dairy cattle breeds: German Holstein (HOL), Angler (ANG), and Red and White Dual-Purpose (RDN). For that purpose, the stochastic bio-economic model SimHerd (SimHerd A/S, Viborg, Denmark) was used, which simulates the expected monetary gain in dairy herds. The EV was calculated as the alteration in average net return of the herd responding to a marginal change in the trait of interest. When deriving EV using SimHerd, economic consequences resulting from changes in the age structure of a dairy herd (i.e., structural herd effects) are considered. However, this requires the simulation of relationships between traits in the bio-economic model. To avoid double counting, the EV of a trait was corrected for effects from alterations in correlated traits using multiple regression analysis. The EV were derived for 23 traits in terms of production, conformation and workability, dairy health, calf survival, and reproduction performance. Furthermore, the relative economic importance of the breeding traits was calculated. Relative emphasis on production was between 39.9 and 44.4% in the breeds studied. Total costs per case of ketosis and metritis ranged from €167 to €196 and €173 to €182, respectively. Highest marginal EV of direct health traits were found for mastitis (€257 to €271 per case) and lameness (€270 to €310 per case). Consequently, relative emphasis on direct health traits was between 15.7 and 17.9%. The EV of reproduction performance showed largest differences among the cattle breeds. Overall relative emphasis on reproduction was 10.5% in HOL, 10.8% in ANG, and 6.5% in RDN. The relative economic importance of cow mortality ranged from 15.5 to 16.0% across the breeds. Collectively, the study showed the high economic importance of functional traits in the cattle breeds studied.

Surgical publication activity in the English literature over a 10-year interval.

Background: Surgical publication activity in the English literature over a 10-year interval may have changed. This study sought to identify which countries make the most contributions and whether significant shifts have occurred in this time. Methods: Screening of 17 international journals in PubMed was performed for the time periods 2006-2007 and 2016-2017, for papers published by a first author belonging to a general surgical department. Data were collected by country regarding the total number of publications, cumulative impact factors (IFs), publications per inhabitant, IFs per inhabitant, and number of RCTs, meta-analyses and systematic reviews per country in both periods. Results: A total of 2247 and 3029 papers were found for 2006-2007 and 2016-2017 respectively. In 2006-2007, most papers (605, 26·9 per cent; 2697·3 IFs) came from the USA, followed by Japan (284, 12·6 per cent; 1042·1 IFs) and the UK (197, 8·8 per cent; 923·1 IFs). In 2016-2017, the USA led again with 898 papers (29·6 per cent; 4575·3 IFs), followed by Japan with 414 papers (13·7 per cent; 1556·6 IFs) and the Netherlands with 167 (5·5 per cent; 885·2 IFs). From the top 15 countries, Sweden, the Netherlands and Switzerland contributed the most articles per inhabitant during both time periods. During both periods, the UK published the most RCTs, meta-analyses and systematic reviews. Conclusion: Surgeons from the USA were the most productive in total number of publications during both time periods. However, smaller European countries were more active than the USA in relation to their population size.

Exploration of conservation and development strategies with a limited stakeholder approach for local cattle breeds.

Many local breeds have become endangered due to their substitution by high-yielding breeds. To conserve local breeds, effective development strategies need to be investigated. The aim of this study was to explore conservation and development strategies based on quantified strengths, weaknesses, opportunities and threats (SWOT) for two local cattle breeds from Northern Germany, namely the German Angler (GA) and Red Dual-Purpose cattle (RDP). The data comprised 158 questionnaires regarding both breeds' SWOT, which were answered by 78 farmers of GA and 80 farmers of RDP. First, data were analysed using the SWOT-Analytic Hierarchy Process (AHP) method, which combines the qualitative strategic decision tool of SWOT analysis and the quantitative tool of AHP. Second, prioritised SWOT factors were discussed with stakeholders in order to form final conservation and development strategies at breed level. For GA prioritised strengths were daily gain, meat quality, milk production and the usage of new biotechnologies, weaknesses were genetic gain in milk production and inbreeding, opportunities were organic farming and breed-specific characteristics and threats were milk prices and dependency regarding the dairy business. Consequently, three conservation and development strategies were formed: (1) changing relative weights and the relevant breeding goal to drift from milk to meat, (2) increasing genetic gain and control the rate of inbreeding by the implementation of specific selection programs and (3) selection of unique and breed characteristic components on product level, that is, milk-fat and fine muscle fibers. For RDP defined strengths were robustness, high adaptability for different housing systems and a balanced dual-purpose of milk and meat, weaknesses were inbreeding, breed extinction, genomic selection with young bulls and milk yield, opportunities were organic farming and dual-purpose aspects and threats were milk and decreasing beef cattle prices. Thus, three conservation and development strategies were identified: (1) adjust relative weights and the relevant breeding goal to balance milk and meat yield, (2) increasing genetic gain and avoid extinction by implementing targeted selection programs and (3) selection of unique and breed characteristic traits on breed level, that is, environmental robustness. Quantified SWOT establish a basis for the exploration of conservation and development strategies at breed level. Explored strategies are promising even if the stakeholder approach was limited for small populations regarding a small number of stakeholder groups. The used approach reflects farmers' individual convenience better than existing quantitative strategy decision tools on their own.

The size-selective interaction of key and lock nanocrystals driven by depletion attraction at the nanoscale.

In this article, we study the size-dependent interactions of quasi-spherical nanocrystals with voids of concave nanoparticles of complementary sizes and shapes. Experimental insights into a system with key and lock particles with smaller dimensions than 15 nm are presented, which provide evidence for key-lock specific interaction on this length scale. Using depletion attraction as a driving force, the key-lock interaction is shown to be reversible and independent of the material composition of the key particles. Poly(ethylene glycol) methacrylate was utilized as a depletion agent in toluene, the solvent of the studied key-lock system. For this work, a model system of specifically developed concave manganese oxide nanocrystals, synthesized via a cast-mold approach, in combination with highly monodisperse quasi-spherical gold nanocrystals, was investigated with transmission electron microscopy, optical UV/vis/NIR spectroscopy and powder X-ray diffraction. Size-dependent key-lock interactions are clearly identified to occur. For geometrical reasons, only key particles with smaller particle diameters than the voids of the complementary lock particles are able to enter the void. So the void diameter of the lock particles sets a diameter threshold for the key-lock interaction. Additionally, other key particles like silver, iron oxide and even core-shell structured gold-nickel sulfide nanocrystals show key-in-lock assemblies with concave manganese oxide nanocrystals. This behaviour might open up new routes for size-selective particle sensing.

Genome-wide mapping of 10 calving and fertility traits in Holstein dairy cattle with special regard to chromosome 18.

Over the last decades, a dramatic decrease in reproductive performance has been observed in Holstein cattle and fertility problems have become the most common reason for a cow to leave the herd. The premature removal of animals with high breeding values results in both economic and breeding losses. For efficient future Holstein breeding, the identification of loci associated with low fertility is of major interest and thus constitutes the aim of this study. To reach this aim, a genome-wide combined linkage disequilibrium and linkage analysis (cLDLA) was conducted using data on the following 10 calving and fertility traits in the form of estimated breeding values: days from first service to conception of heifers and cows, nonreturn rate on d 56 of heifers and cows, days from calving to first insemination, days open, paternal and maternal calving ease, paternal and maternal stillbirth. The animal data set contained 2,527 daughter-proven Holstein bulls from Germany that were genotyped with Illumina's BovineSNP50 BeadChip (Illumina Inc., San Diego, CA). For the cLDLA, 41,635 sliding windows of 40 adjacent single nucleotide polymorphisms (SNP) were used. At each window midpoint, a variance component analysis was executed using ASReml. The underlying mixed linear model included random quantitative trait locus (QTL) and polygenic effects. We identified 50 genome-wide significant QTL. The most significant peak was detected for direct calving ease at 59,179,424 bp on chromosome 18 (BTA18). Next, a mixed-linear model association (MLMA) analysis was conducted. A comparison of the cLDLA and MLMA results with special regard to BTA18 showed that the genome-wide most significant SNP from the MLMA was associated with the same trait and located on the same chromosome at 57,589,121 bp (i.e., about 1.5 Mb apart from the cLDLA peak). The results of 5 different cLDLA and 2 MLMA models, which included the fixed effects of either SNP or haplotypes, suggested that the cLDLA method outperformed the MLMA in accuracy and precision. The haplotype-based cLDLA method allowed for a more precise mapping and the definition of ancestral and derived QTL alleles, both of which are essential for the detection of underlying quantitative trait nucleotides.

Estimation of ancestral inbreeding effects on stillbirth, calving ease and birthweight in German Holstein dairy cattle.

In this study, the effect of different measurements of ancestral inbreeding on birthweight, calving ease and stillbirth were analysed. Three models were used to estimate the effect of ancestral inbreeding, and the estimated regression coefficient of phenotypic data on different measurements of ancestral inbreeding was used to quantify the effect of ancestral inbreeding. The first model included only one measurement of inbreeding, whereas the second model included the classical inbreeding coefficients and one alternative inbreeding coefficient. The third model included the classical inbreeding coefficients, the interaction between classical inbreeding and ancestral inbreeding, and the classical inbreeding coefficients of the dam. Phenotypic data for this study were collected from February 1998 to December 2008 on three large commercial milk farms. During this time, 36,477 calving events were recorded. All calves were weighed after birth, and 8.08% of the calves died within 48 h after calving. Calving ease was recorded on a scale between 1 and 4 (1 = easy birth, 4 = surgery), and 69.95, 20.91, 8.92 and 0.21% of the calvings were scored with 1, 2, 3 and 4, respectively. The average inbreeding coefficient of inbred animals was 0.03, and average ancestral inbreeding coefficients were 0.08 and 0.01, depending on how ancestral inbreeding was calculated. Approximately 26% of classically non-inbred animals showed ancestral inbreeding. Correlations between different inbreeding coefficients ranged between 0.46 and 0.99. No significant effect of ancestral inbreeding was found for calving ease, because the number of animals with reasonable high level of ancestral inbreeding was too low. Significant effects of ancestral inbreeding were estimated for birthweight and stillbirth. Unfavourable effects of ancestral inbreeding were observed for birthweight. However, favourable purging effects were estimated for stillbirth, indicating that purging could be partly beneficial for genetic improvement of stillbirth.

Estimation of variance components for postpartum dysgalactia syndrome in sows.

The postpartum dysgalactia syndrome (PDS) represents one of the most important diseases after parturition in sows. The genetic background of the disease has been investigated some time ago and heritability estimates around 0.10 have been obtained. To compute current estimates, a dataset of 1680 sampled sows and their 2001 clinically examined litters was used for variance components estimation with a threshold liability model. Affected sows were defined through clinical examination 12-48 h after parturition. Posterior mean of additive genetic variance was 0.10 and estimated heritability for PDS averaged 0.0879 with a 95% confidence interval of 0.0876 and 0.0881. The results are in agreement with those of other studies and emphasize the importance of considering the genetic predisposition for susceptibility to PDS as well as of additional factors including hygiene and management conditions.

Pedigree analysis and inbreeding effects on calving traits in large dairy herds in Germany.

A pedigree analysis was carried out for a subset of the German Holstein population with respect to the effective number of founders and the ancestors with the highest effect on 2 defined reference populations. Reference population 1 contained animals born between 1998 and 2002 (n=19,537), and reference population 2 included animals born between 2003 and 2007 (n=19,060). The pedigree file included 73,946 animals in total. The effective number of founders was 111.3 and 92.8 in reference populations 1 and 2, respectively. In reference population 1, 52.22% of the gene pool could be explained by 10 ancestors. In reference population 2, the 10 ancestors with the greatest effect contributed 57.22% of the gene pool. The effect of inbreeding on birth weight, calving ease, and stillbirth was also analyzed in this study. Two models were used to estimate the effect of inbreeding. One model fitted inbreeding as a regression on the inbreeding coefficient of the dam, whereas the other model fitted inbreeding as a regression on the inbreeding coefficient of the calf. Phenotypic data for this study were collected on 3 large commercial milk farms. Data recording took place from February 1998 to December 2008 and 36,623 calving events were recorded; 8.19% of the calves died at or within 48 h after calving. All calves were weighed after birth and the average birth weight was 43 kg for calves born alive and 42 kg for stillborn calves. Calving ease was recorded on a scale between 1 and 4. The distribution of scores was 69.81, 21.02, 8.96, and 0.22%, ranked 1 (without assistance or assistance by 1 person), 2 (assistance by 2 or more persons or mechanical assistance), 3 (veterinary assistance), and 4 (operation), respectively, on the calving-ease scale. Twins were excluded from all analyses. Dam inbreeding had no significant effect on the traits of this study. The inbreeding coefficient of the calf had no significant effect on birth weight and calving ease but showed a significant effect on stillbirth: the risk of stillbirth was found to increase by 0.22% per 1% increase of the inbreeding coefficient of the calf.

Analyzing the effect of different approaches of penalized relationship in multistage selection schemes.

The aim of this study was to extend optimum contribution selection to more realistic breeding schemes with multistage selection. It seems that if the last selection stage accounts for the relationship of the selected animals, then previous selection stages also account for this relationship. An extreme example was considered here: the preselection of dairy bulls that enter a progeny testing scheme. First the penalty on the average relationship in selection step 1 is assumed the same as in step 2. Thereafter, situations with different penalties on the average relationship in the 2 selection steps were analyzed. The simulation started with the generation of prior EBV, which were sampled from a truncated normal distribution. Possible candidates for further progeny testing were selected and progeny test EBV were simulated, where the progeny test was based on 100 daughters per young bull. In situations with greater accuracy of prior EBV, high trait heritability and prior EBV were available for 2,000 bulls; the results were similar for both approaches, independent of family size. However, in a situation with low accuracy of prior EBV and low trait heritability it could be observed that with increasing penalty on the average relationship, correction for relationship in stage 1 yielded in a similar genetic level compared with selecting only for high prior EBV. If the number of bulls with prior EBV increased from 2,000 to 4,000, an increasing penalty on an average relationship gave an improved genetic level. A further improvement of the results with respect to genetic level and average relationship could be observed by increasing the penalty on an average relationship in selection step 1 above that in selection step 2. Overall, this study showed that it is beneficial to use a penalty on an average relationship already for the selection of bulls that enter the progeny test. In case optimum contribution was applied with a constraint on the average relationship in stage 2, this constraint may be translated into a penalty on the average relationship, and the current results suggested that the optimal penalty in selection stage 1 should be twice that of stage 2.

Genetic analysis of mastitis data with different models.

The aim of this study was to analyze different mastitis data sets with different statistical models and compare results. Data recording took place on 3 commercial milk farms with an average herd size of 3,200 German Holstein cows. Recording started in February 1998 and was completed in December 2005. During this period, 63,540 treatments for clinical mastitis were recorded. Five different data sets were analyzed and the number of cows varied between 12,972 and 13,618, depending on the data set. Data collection periods contained either the first 50 or the first 300 d of lactation. When the data-recording period ended after 50 d of lactation, data sets were analyzed with a lactation threshold model (LTM), a multiple threshold lactation model (MTLM), and a test-day threshold model (TDTM). In the LTM analysis, mastitis was treated as a binary trait coded as 0 (no mastitis) or 1 (mastitis), whereas in MTLM mastitis, codes were between 0 and 4, depending on the number of estimated days with mastitis. The TDTM treated each day as a single observation coded similarly to that of the LTM. When the data collection period included the first 300 d of lactation, data sets were analyzed with the LTM or MTLM only, because the TDTM was computationally infeasible. Mastitis frequencies in LTM data sets were 25.8 and 39.2%, and 26.9 and 39.2% in MTLM data sets, when data recording ended after 50 and 300 d of lactation, respectively. The mastitis frequency in the TDTM data set was 5.2%. Respective heritability estimates of liability to clinical mastitis were 0.08 and 0.09 using the LTM, and 0.08 and 0.11 using the MTLM. When the TDTM was used, the estimated heritability was 0.15. Rank correlation between breeding values of the different data sets ranged between 0.40 and 0.97. Rank correlation between the LTM and MTLM were higher (0.78 to 0.97) than those between these 2 models and the TDTM (0.40 to 0.59).The MTLM combined the positive effects of both the LTM, with respect to the size of the data sets, and the TDTM, with respect to the lack of information.

Analysis of inbreeding depression in the first litter size of mice in a long-term selection experiment with respect to the age of the inbreeding.

An understanding of inbreeding and inbreeding depression are important in evolutionary biology, conservation genetics, and animal breeding. A new method was developed to detect departures from the classical model of inbreeding; in particular, it investigated differences between the effects of inbreeding in recent generations from that in the more distant past. The method was applied in a long-term selection experiment on first-litter size in mice. The total pedigree included 74,630 animals with approximately 30,000 phenotypic records. The experiment comprised several different lines. The highest inbreeding coefficients (F) within a line ranged from 0.22 to 0.64, and the average effective population size (N(e)) was 58.1. The analysis divided F into two parts, corresponding to the inbreeding occurring in recent generations ('new') and that which preceded it ('old'). The analysis was repeated for different definitions of 'old' and 'new', depending on length of the 'new' period. In 15 of these tests, 'new' inbreeding was estimated to cause greater depression than 'old'. The estimated depression ranged from -11.53 to -0.79 for the 'new' inbreeding and from -5.22 to 15.51 for 'old'. The difference was significant, the 'new' period included at least 25 generations of inbreeding. Since there were only small differences in N(e) between lines, and near constant N(e) within lines, the effect of 'new' and 'old' cannot be attributed to the effects of 'fast' versus 'slow' inbreeding. It was concluded that this departure from the classical model, which predicts no distinction between this 'old and 'new' inbreeding, must implicate natural selection and purging in influencing the magnitude of depression.

An algorithm to compute optimal genetic contributions in selection programs with large numbers of candidates.

A novel algorithm, OCSELECT, is presented for the calculation of optimal genetic contributions with a restricted rate of inbreeding when the number of selection candidates is very large. The calculation of optimal genetic contributions requires the relationship matrix between the candidates and its inverse. The relationship matrix was written as: A = ZA(p)Z' + D, where A(p) is the relationship matrix of the parents, D is a diagonal matrix of Mendelian sampling variances, and Z contains genetic contributions from parents to offspring. Therefore, A(-1) = d(-1) - d(-1)Z(Z'd(-1)Z + A(P)(-1))(-1) Z'd(-1), requires only inversion of matrices of the size of the number of parents instead of the number of offspring. The new algorithm was compared with the software package GENCONT on a salmon data set containing 39,214 selection candidates and 45,846 pedigreed fish in total. Because GENCONT could not handle such a large data set, this data set was split into 19 smaller data sets. Both algorithms gave the same solution with respect to the genetic gain and very similar solutions with respect to the number of selected animals. The OCSELECT algorithm was able to calculate the optimal contributions for the complete data set of 39,214, and therefore no preselection of the 39,214 fish was necessary before entering the fish into the new optimal contribution selection procedure.

Genetic parameters for health traits and their relationship to different persistency traits in German Holstein dairy cattle.

Data from 3,200 Holstein cows from 3 commercial dairy farms in Germany were used to estimate heritabilities and breeding values for liability to udder diseases (UD), fertility diseases (FD), metabolic diseases (MD), and claw and leg diseases (CLD) using single-trait threshold sire models. A total of 92,722 medical treatments recorded from 1998 to 2003 were included in the analysis. Approximate genetic correlations between persistency of milk yield, fat yield, protein yield, and persistency of milk energy yield and liability to the health traits were calculated based on correlations between EBV. Posterior means of heritability of liability ranged from 0.05 to 0.08 for UD, from 0.04 to 0.07 for FD, from 0.08 to 0.12 for MD, and from 0.04 to 0.07 for CLD. Approximate genetic correlations of the disease traits with the persistency traits were favorable, except for MD in all lactations, which were unfavorable, and UD, which were around zero. Highest correlations in the range of 0.13 to 0.46 were found between the different persistency traits and CLD.

Genetic analyses of mastitis data using animal threshold models and genetic correlation with production traits.

In the present study, 6 different mastitis data sets of 3 dairy herds with an overall herd size of 3200 German Holstein cows were analyzed. Data collection periods included the first 50, 100, or 300 d of lactation. The 3 data collection periods were analyzed with a lactation model and a test-day model. All models were animal threshold models. Mastitis frequencies in the lactation model data sets varied between 29 and 45%, and varied between 3 and 6% in the test-day model data sets. Depending on the period of data collection, heritabilities of liability to mastitis in the lactation models were 0.05 (50 d), 0.06 (100 d), and 0.07 (300 d). In the test-day models, heritabilities were slightly higher with values of 0.09 (50 and 100 d), and 0.06 (300 d). Between lactation models, the rank correlations between the relative breeding values were high and varied between 0.86 and 0.94. Rank correlations between the relative breeding values of the test-day models ranged from 0.68 to 0.87. The rank correlations between the relative breeding values of lactation models and test-day models varied from 0.51 and 0.80. Genetic correlations between mastitis and milk production traits were estimated with a linear animal test-day model. The correlations with mastitis were 0.29 (milk yield), 0.30 (fat yield), 0.20 (fat content), 0.34 (protein yield), and 0.20 (protein content). The estimated genetic correlation between mastitis and somatic cell score was 0.84.

Quantitative determination of plasma c8-c26 total fatty acids for the biochemical diagnosis of nutritional and metabolic disorders.

We have developed a capillary gas chromatography-electron-capture negative-ion mass spectrometry (GC/MS) method for the quantitative determination of C8-C26 total fatty acids in plasma. Following hydrolysis, hexane extraction, and derivatization with pentafluorobenzyl bromide, fatty acid esters are analyzed in two steps: a splitless injection and a second, split injection (1:100) for the quantitation of the more abundant long-chain species. Fourteen saturated and 25 unsaturated fatty acids are quantified by selected ion monitoring in ratio to 13 stable-isotope-labeled internal standards. Calibrations exhibit consistent linearity and reproducibility. Intraassay (n = 17) and interassay (n = 12) CVs ranged from 2.5 to 13.2% and from 4.6 to 22.9%, respectively. Recoveries ranged from 76 to 106%. Reference ranges were established for four age groups (<1 month, 1 month to 1 year, 1-17 years, >18 years) and compared to specimens from patients with nutritional deficiency of omega-3 and omega-6 polyunsaturated fatty acids, inborn errors of mitochondrial fatty acid oxidation, and peroxisomal disorders. Retrospective evaluation of the concentration of linoleic acid in 35 cases with a diagnosis of essential fatty acid deficiency previously made by gas chromatographic analysis with flame ionization detection (GC/FID) found a specificity and sensitivity of only 55 and 50%, respectively, for the GC/FID method when compared to GC/MS.

Lack of expansion of major histocompatibility complex class Ib-restricted effector cells following recovery from secondary infection with the intracellular pathogen Listeria monocytogenes.

Sublethal infection of BALB/c mice with the intracellular bacterial pathogen Listeria monocytogenes leads to the development of antilisterial immunity with concurrent stimulation of major histocompatibility complex (MHC) class Ia- and Ib-restricted CD8+ effector T cells. Secondary L. monocytogenes infection is followed by an accelerated increase in the number of Listeria-specific CD8+ cells and rapid clearance of the bacterium from the murine host. Recovery from secondary infection is associated with increased levels of effector cell function, as measured by gamma interferon secretion following coculture of immune cells with L. monocytogenes infected APCs in vitro, as well as antilisterial cytotoxicity, as measured by effector cell recognition of L. monocytogenes-infected target cells. We assessed the frequency of L. monocytogenes-specific MHC class I-restricted cells following secondary infection by ELISPOT assays utilizing coculture of immune cells with L. monocytogenes-infected antigen-presenting cells that express MHC class Ia and/or Ib molecules. We found that the antilisterial Qa-1b (MHC class Ib)-restricted effector subset is not detected as an expanded population following secondary infection compared to the frequency of this effector population as measured following recovery from primary infection. This is in contrast to the frequency of antilisterial H2-Kd (MHC class Ia)-restricted effector cells, which following recovery from secondary infection are detected as an expanded population, and appears to undergo a substantial expansion event 3 to 4 days post-secondary infection. These results are consistent with the conclusion that although Listeria-specific MHC class Ib-restricted effector cells are present following recovery from secondary infection, this subset does not appear to undergo the expansion phase that is detected for the MHC class Ia-restricted effector cell response.

Association of multispecific CD4(+) response to hepatitis C and severity of recurrence after liver transplantation.

BACKGROUND & AIMS: After liver transplantation for hepatitis C virus (HCV), reinfection of the allograft invariably occurs. Indirect evidence suggests that the cellular immune response may play a central role. The purpose of this analysis was to determine the correlation between HCV-specific peripheral CD4(+) T-cell responses and the severity of recurrence after liver transplantation. METHODS: Fifty-eight HCV-seropositive patients, including 43 liver transplant recipients with at least 1 year of histological follow-up, were studied. Peripheral blood mononuclear cells (PBMCs) were isolated from fresh heparinized blood and stimulated with either recombinant HCV antigens (core, E2, NS3, NS4, and NS5) or control antigens. RESULTS: Fourteen (40%) of 35 patients with mild or no evidence of histological recurrence within their allografts responded to at least 1 of the HCV antigens. Eleven responded to NS3, 5 to all the nonstructural antigens, and 3 to the HCV core polypeptide alone. In contrast, in the 8 patients with severe HCV recurrence, no proliferation in response to any of the HCV antigens was seen (P = 0. 03) despite responses to the control antigens. CONCLUSIONS: Despite immunosuppression, HCV-specific, major histocompatibility complex class II- restricted CD4(+) T-cell responses are detectable in patients with minimal histological recurrence after liver transplantation. In contrast, PBMCs from patients with severe HCV recurrence, despite being able to proliferate in response to non-HCV antigens, fail to respond to the HCV antigens. These findings suggest that the inability to generate virus-specific T-cell responses plays a contributory role in the pathogenesis of HCV-related graft injury after liver transplantation. It is hoped that further characterization of the immunoregulatory mechanisms related to recurrent HCV will provide the rationale for novel therapeutic strategies and diminish the incidence of inevitable graft loss.

Genetic immunization of mice against Listeria monocytogenes using plasmid DNA encoding listeriolysin O.

The development of protective immunity against many intracellular bacterial pathogens commonly requires sublethal infection with viable forms of the bacteria. Such infection results in the in vivo activation of specific cell-mediated immune responses, and both CD4+ and CD8+ T lymphocytes may function in the induction of this protective immunity. In rodent models of experimental infection with Listeria monocytogenes, the expression of protective immunity can be mediated solely by the immune CD8+ T cell subset. One major target Ag of Listeria-immune CD8+ T cells is the secreted bacterial hemolysin, listeriolysin O (LLO). In an attempt to generate a subunit vaccine in this experimental disease model, eukaryotic plasmid DNA expression vectors containing genes encoding either the wild-type or modified forms of recombinant LLO were generated and used for genetic vaccination of naive mice. Results of these studies indicate that the intramuscular immunization of mice with specifically designed plasmid DNA constructs encoding recombinant forms of LLO stimulates peptide-specific CD8+ immune T cells that exhibit in vitro cytotoxic activity. More importantly, such immunization can provide protective immunity against a subsequent challenge with viable L. monocytogenes, demonstrating that this experimental approach may have direct application in prevention of acute disease caused by intracellular bacterial pathogens.

Existing antilisterial immunity does not inhibit the development of a Listeria monocytogenes-specific primary cytotoxic T-lymphocyte response.

Infection of BALB/c mice with Listeria monocytogenes stimulates an antilisterial immune response evident by the appearance of H2-Kd-restricted CD8(+) cytotoxic T lymphocytes (CTLs) specific for the nanomer peptides amino acids (aa) 91 to 99 of listeriolysin O (LLO 91-99) and aa 217 to 225 of the p60 molecule (p60 217-225). We have introduced point mutations at anchor residues within LLO 91-99 (92F) or p60 217-225 (218F), and BALB/c mice infected with L. monocytogenes strains containing these point mutations do not develop CTLs specific for LLO 91-99 or p60 217-225, respectively. We have used these strains to test whether primary CTL responses against L. monocytogenes-derived determinants can be stimulated within an environment of existing antilisterial immunity. We found that the development of a primary L. monocytogenes-specific CTL response is not altered by existing immunity to L. monocytogenes. For example, primary immunization with the p60 218F strain of L. monocytogenes followed by a secondary immunization with wild-type L. monocytogenes results in stimulation of p60 217-225-specific CTLs at primary response levels and LLO 91-99-specific effectors at levels consistent with a memory CTL response. Similarly, primary immunization with the 92F strain of L. monocytogenes followed by a secondary immunization with wild-type L. monocytogenes results in stimulation of LLO 91-99-specific CTLs at primary response levels and p60 217-225-specific effectors at levels consistent with a memory CTL response. These results provide additional support for the use of L. monocytogenes as a recombinant vaccine vector and show that antivector immunity does not inhibit the development of a primary CTL response when the epitope is delivered by L. monocytogenes as the vaccine strain.