Effects of trace minerals source in the broiler breeder diet and eggshell translucency on embryonic development of the offspring.

In 2 experiments, interactions between trace mineral (Zn, Mn, Cu, Se) source (organic or inorganic) in the broiler breeder diet and egg translucency (high or low) on egg characteristics and embryonic development were investigated. In the first experiment, eggs from old breeders (55-57 wk) and in the second experiment, eggs from prime breeders (34-36 wk) were used. Egg composition and bacterial load on the eggshell were analyzed in fresh eggs. During incubation, metabolic heat production of the embryos (d 8 (E8) to 19 of incubation) and tibia ossification (E8.5-E14.5) were determined daily. At hatch, chicken quality was assessed, including tibia biophysical characteristic. Egg quality was not affected by breeder trace minerals source or egg translucency in both experiments. In both experiments, an interaction between trace minerals source and translucency score was found for egg weight loss during incubation. In inorganic trace minerals fed breeders, a high egg translucency resulted in a higher egg weight loss than a low egg translucency, whereas this difference was not seen in organic trace minerals fed breeders. Embryonic heat production and tibia ossification were not affected by trace minerals source or egg translucency. Chicken quality showed ambiguous results between experiment 1 and 2 regarding trace minerals source in the breeder diet. In experiment 2, high translucent eggs from organic fed breeders hatched later than eggs from the other three treatment groups and additionally, high egg translucency resulted in lower residual yolk weight and higher heart and liver percentage of YFBM compared to low egg translucency. Tibia biophysical characteristics at hatch were not affected by trace minerals source or egg translucency. It can be concluded that organic trace minerals source in broiler breeder diet affects eggshell conductance, particularly in low translucent eggs, but effects on chicken quality and tibia characteristics appears to be limited.

Gut microbiota of homing pigeons shows summer-winter variation under constant diet indicating a substantial effect of temperature.

BACKGROUND: Gut microbiotas play a pivotal role in host physiology and behaviour, and may affect host life-history traits such as seasonal variation in host phenotypic state. Generally, seasonal gut microbiota variation is attributed to seasonal diet variation. However, seasonal temperature and day length variation may also drive gut microbiota variation. We investigated summer-winter differences in the gut bacterial community (GBC) in 14 homing pigeons living outdoors under a constant diet by collecting cloacal swabs in both seasons during two years. Because temperature effects may be mediated by host metabolism, we determined basal metabolic rate (BMR) and body mass. Immune competence is influenced by day length and has a close relationship with the GBC, and it may thus be a link between day length and gut microbiota. Therefore, we measured seven innate immune indices. We expected the GBC to show summer-winter differences and to correlate with metabolism and immune indices. RESULTS: BMR, body mass, and two immune indices varied seasonally, other host factors did not. The GBC showed differences between seasons and sexes, and correlated with metabolism and immune indices. The most abundant genus (Lachnoclostridium 12, 12%) and associated higher taxa, were more abundant in winter, though not significantly at the phylum level, Firmicutes. Bacteroidetes were more abundant in summer. The Firmicutes:Bacteroidetes ratio tended to be higher in winter. The KEGG ortholog functions for fatty acid biosynthesis and linoleic acid metabolism (PICRUSt2) had increased abundances in winter. CONCLUSIONS: The GBC of homing pigeons varied seasonally, even under a constant diet. The correlations between immune indices and the GBC did not involve consistently specific immune indices and included only one of the two immune indices that showed seasonal differences, suggesting that immune competence may be an unlikely link between day length and the GBC. The correlations between the GBC and metabolism indices, the higher Firmicutes:Bacteroidetes ratio in winter, and the resemblance of the summer-winter differences in the GBC with the general temperature effects on the GBC in the literature, suggest that temperature partly drove the summer-winter differences in the GBC in homing pigeons.

Genetic parameters of colostrum and calf serum antibodies in Swedish dairy cattle.

BACKGROUND: A sufficient IgG content in the colostrum is essential for the newborn calf, as it provides passive immunity which substantially affects the probability of survival during rearing. Failure of passive transfer (FPT) occurs when a calf does not absorb enough antibodies from the colostrum and is defined by an IgG concentration in calf serum lower than 10 g/L. Apart from delayed access to colostrum, FPT can be due to a low production of IgG in the mother or poor IgG absorption by the calf. The aim of this study was to estimate the genetic background of antibody levels and indicator traits for antibodies in the colostrum and calf serum, and their correlation with milk production. RESULTS: Colostrum data were available for 1340 dairy cows with at least one calving and calf serum data were available for 886 calves from these cows. Indicator traits for antibody concentrations were estimated using refractometry (a digital Brix refractometer for colostrum and an optical refractometer for serum), and enzyme-linked immunosorbent assays (ELISA) were used to determine the levels of total IgG and natural antibodies (NAb) of various antibody isotypes in the colostrum and calf serum. Colostrum traits had heritabilities ranging from 0.16 to 0.31 with repeatabilities ranging from 0.21 to 0.55. Brix percentages had positive genetic correlations with all colostrum antibody traits including total IgG (0.68). Calf serum antibody concentrations had heritabilities ranging from 0.25 to 0.59, with a significant maternal effect accounting for 17 to 27% of the variance. When later in life calves produced their first lactation, the lactation average somatic cell score was found to be negatively correlated with NAb levels in calf serum. CONCLUSIONS: Our results suggest that antibody levels in the colostrum and calf serum can be increased by means of selection.

Transgenerational Effects of Maternal Immune Activation on Specific Antibody Responses in Layer Chickens.

Activation of the maternal immune system may affect innate and adaptive immune responses in the next generation and may therefore have implications for vaccine efficacy and dietary immune modulation by feed additives. However, transgenerational effects on immune responses in chickens have been investigated to a limited extend. The present study investigated effects of intratracheal (i.t) specific and aspecific immune activation of laying hens on specific antibody production in the next generation. In two experiments laying hens received intratracheally an immune stimulus with human serum albumin (HuSA) or lipopolysaccharide (LPS). In experiment 1, hatchlings of the immune activated hens were at 4 weeks i.t. immunized with HuSA or HuSA+LPS. Maternal immune activation with LPS increased HuSA specific IgY and IgM responses in offspring. These results suggest a transgenerational effect of the maternal immune system on the specific antibody response in the next generation. In experiment 2 hatchlings received either ß-glucan-enriched feed or control feed and were i.t. immunized with HuSA. Maternal immune activation with LPS decreased IgY anti-HuSA responses after HuSA immunization within hatchlings that received ß-glucan enriched feed. The results of Experiment 2 suggest a transgenerational link between the innate immune system of mother and specific antibody responses in offspring. Despite variabilities in the outcomes of the two experiments, the observations of both suggest a link between the maternal innate immune system and the immune system of the offspring. Furthermore, our results may imply that maternal activation of the innate immune system can influence immune modulating dietary interventions and vaccine strategies in the next generation.

Divergent selection for natural antibodies in poultry in the presence of a major gene.

BACKGROUND: Natural antibodies (NAb) are antibodies that are present in a healthy individual without requiring previous exposure to an exogenous antigen. Selection for high NAb levels might contribute to improved general disease resistance. Our aim was to analyse the genetic background of NAb based on a divergent selection experiment in poultry, and in particular the effect of a polymorphism in the TLR1A gene. METHODS: The study population consisted of a base population from a commercial pure-bred elite white leghorn layer line and seven generations of birds from a High and Low selection line. Birds were selected for total KLH-binding NAb titer (IgTotal). An enzyme-linked immunosorbent assay was performed to determine NAb titers in blood plasma for IgTotal and the antibody isotypes IgM and IgG. NAb titers were available for 10,878 birds. Genotypes for a polymorphism in TLR1A were determined for chickens in generations 5, 6 and 7. The data were analysed using mixed linear animal models. RESULTS: The heritability estimate for IgM was 0.30 and higher than that for IgG and IgTotal (0.12). Maternal environmental effects explained 2 to 3% of the phenotypic variation in NAb. Selection for IgTotal resulted in a genetic difference between the High and Low line of 2.4 titer points (5.1 genetic standard deviation) in generation 7. For IgM, the selection response was asymmetrical and higher in the Low than the High line. The frequency of the TLR1A C allele was 0.45 in the base population and 0.66 and 0.04 in generation 7 of the High and Low line, respectively. The TLR1A polymorphism had large and significant effects on IgTotal and IgM. Estimated genotypic effects suggest full dominance of the TLR1A C allele. Significant TLR1A by generation interactions were detected for IgM and IgTotal. CONCLUSIONS: The effect of a polymorphism in the TLR1A gene on IgTotal and IgM NAb was confirmed. Furthermore, we provide experimental verification of changes in allele frequencies at a major gene with dominant gene action on a quantitative trait that is subjected to mass selection. TLR1A by generation interactions indicate sensitivity to environmental factors.

Innate immune training and metabolic reprogramming in primary monocytes of broiler and laying hens.

Recently, we have reported trained innate immunity in laying chicken monocytes. In the present study, we further investigated trained innate immunity of monocytes in layers and broilers. Monocytes of both breeds isolated from blood were trained in vitro with ß-glucan, rec-chicken IL-4 or a combination of both, and restimulated with lipopolysaccharide (LPS), after which inflammation and metabolism-related responses were measured. Training of laying and broiler hen monocytes resulted in increased mRNA levels of IL-1ß, iNOS and HIF-1α, but enhanced surface expression of CD40 and NO production was only observed in layers. Our in vitro study demonstrates that monocytes from different genetic backgrounds can be trained. However, the observed differences suggest a differential effect on immune functionality associated with innate training. Whether these differences in immune functions between layers and broilers have effect on disease resistance remains to be elucidated.

Early and later life environmental enrichment affect specific antibody responses and blood leukocyte subpopulations in pigs.

This study addressed the impact of early and later life environmental enrichment, and their combination, on specific antibody responses and peripheral blood leukocyte subpopulations in pigs. Pigs were kept in either barren (B1) or enriched (E1) housing from birth, and half of the pigs switched to barren or enriched housing on day 47, resulting in four treatment combinations: B1B2, B1E2, E1B2, E1E2). Pigs were immunized with keyhole limpet hemocyanin-conjugated trinitrophenyl (KLH-TNP) on day 74 and 109 to induce primary and secondary antibody responses. Blood samples were taken weekly until day 130, and IgM and IgG antibody responses were measured. Leukocyte subpopulations were measured on day 74 and 130. Time course of the antibody responses was not affected by housing. Early life enrichment increased the IgG response to KLH, particularly the primary one. At day 74 the relative frequency of lymphocytes, DC and SLA-II expression on monocytes were higher in E1 pigs, whereas the percentage of granulocytes tended to be lower in E1 pigs at day 74. Early life enrichment increased the SLA-II expression on monocytes, the granulocyte to lymphocyte ratio, and tended to increase the percentage of granulocytes, but tended to decrease the percentage of monocytes at day 130. Later life enrichment reduced percentages of CD4+CD8α+ T cells before and after immunization and the SLA-II expression on monocytes at day 74, the percentage of granulocytes and the granulocyte to lymphocyte ratio at day 130. Notably, early and later life housing interacted in their effects on several immune parameters. KLH-IgM responses (both primary and secondary) were affected by the interaction between early and later life housing. IgM titers were higher for B1B2 than for E1E2, with the switched animals (B1E2 and E1B2) moving towards the titers of the animals kept in their later life environment from birth onwards. At day 130 the percentage of gamma delta T cells, CD8α+ cytotoxic T cells and DC were not different between pigs kept in B1B2 and E1E2, but there was a clear impact of the switch in housing conditions, particularly for the pigs that changed from barren to enriched housing. We also found effects of coping style (personality) and sex on some immune parameters. In conclusion, both early life and later life enrichment, and, notably a switch in housing conditions influenced specific antibodies and leukocyte subpopulations in pigs. The current study implies that the early life history of animals and the (mis)match with their current environment could thus be of major importance for their immune system. Further research is needed to investigate potential consequences for the pigs' health.

Genome-wide association study identifies loci influencing natural antibody titers in milk of Dutch Holstein-Friesian cattle.

Natural antibodies (NAb) are produced without any antigenic stimulation as a part of the innate immune system and provide a first line of defense against pathogens. Hence, they may be a useful trait when estimating an animal's potential immune competence and in selection for disease resistance. The aim of this study was to identify genomic regions associated with different NAb traits in milk and potentially describe candidate genes. Milk samples from 1,695 first-lactation Holstein Friesian cows with titer measurements for keyhole limpet hemocyanin, lipopolysaccharide, lipoteichoic acid, and peptidoglycan-binding total NAb and isotypes IgG1, IgM, and IgA were used. Genome-wide association study analyses were performed using imputed 777K SNP genotypes, accounting for relationships using pedigree information. Functional enrichment analysis was performed on the significantly associated genomic regions to look for candidate genes. For IgM NAb, significant associations (false discovery rate <0.05) were found on Bos taurus autosome (BTA) 17, 18, and 21 with candidate genes related to immunoglobulin structure and early B cell development. For IgG1, associations were found on BTA3, and we confirmed a quantitative trait loci on BTA21 previously reported for IgG NAb in serum. Our results provide new insights into the regulation of milk NAb that will help unravel the complex relationship between milk immunoglobulins and disease resistance in dairy cattle.

Maternal Transfer of Natural (Auto-) Antibodies in Chickens.

The presence and relative levels (titers) of IgM and IgG natural antibodies (NAb) binding keyhole limpet hemocyanin (KLH), and natural (auto-) antibodies (N(A)Ab) binding salmon double-stranded DNA (dsDNA), (oxidated-) phosphatidyl (phosphoryl) choline-conjugated bovine serum albumin (PC-BSA), PC-conjugated ovalbumin (PC-OVA), and OVA, respectively, were studied in adult hen plasma, egg yolk, egg albumen, plasma of their hatchlings, and in 8-day-old chick plasma. Birds and eggs were from 2 lines divergently selected for high or low NAb levels binding KLH. This study aimed to determine 1) correlated phenotypic responses of selection for NAb to KLH, 2) transfer of maternal NAb and N(A)Ab via egg compartments, 3) levels of likely maternal NAb and N(A)Ab in hatchlings and 8-day-old chicks, and 4) whether a composite trait: IgM anti-PC-BSA/IgG anti-dsDNA ratio in the compartments could be used as a parameter for health or immune status. NAb and N(A)Ab to all tested antigens were found in adult hens, but low or no levels were found for IgM in yolk and IgG in albumen. Depending on the antigen, NAb and N(A)Ab were found in hatchlings and day 8 birds. Divergent selection and breeding based on NAb binding KLH affected antibody titers of almost all antigens in almost all compartments, in a similar way. Maternal transfer of NAb and N(A)Ab from the adult hen to offspring was via specific routes for specific antigens and isotypes, especially for IgG as suggested by cluster analyses and significant correlations. There was little indication of production of new NAb and N(A)Ab to the studied antigens in either the egg compartments or the hatchlings. A composite trait of IgM PC-BSA/IgG dsDNA ratio was as yet not indicative for immune status, as no significant differences were found between the lines for all compartments. In conclusion, hens provide neonatal chickens with natural (self-) binding IgG antibodies that have been proposed to perform homeostatic functions during the period in which neonates do not produce these antibodies themselves.

Genomic Region Containing Toll-Like Receptor Genes Has a Major Impact on Total IgM Antibodies Including KLH-Binding IgM Natural Antibodies in Chickens.

Natural antibodies (NAb) are antigen binding antibodies present in individuals without a previous exposure to this antigen. Keyhole limpet hemocyanin (KLH)-binding NAb levels were previously associated with survival in chickens. This suggests that selective breeding for KLH-binding NAb may increase survival by means of improved general disease resistance. Genome-wide association studies (GWAS) were performed to identify genes underlying genetic variation in NAb levels. The studied population consisted of 1,628 adolescent layer chickens with observations for titers of KLH-binding NAb of the isotypes IgM, IgA, IgG, the total KLH-binding (IgT) NAb titers, total antibody concentrations of the isotypes IgM, IgA, IgG, and the total antibodies concentration in plasma. GWAS were performed using 57,636 single-nucleotide polymorphisms (SNP). One chromosomal region on chromosome 4 was associated with KLH-binding IgT NAb, and total IgM concentration, and especially with KLH-binding IgM NAb. The region of interest was fine mapped by imputing the region of the study population to whole genome sequence, and subsequently performing an association study using the imputed sequence variants. 16 candidate genes were identified, of which FAM114A1, Toll-like receptor 1 family member B (TLR1B), TLR1A, Krüppel-like factor 3 (KLF3) showed the strongest associations. SNP located in coding regions of the candidate genes were checked for predicted changes in protein functioning. One SNP (at 69,965,939 base pairs) received the maximum impact score from two independent prediction tools, which makes this SNP the most likely causal variant. This SNP is located in TLR1A, which suggests a fundamental role of TLR1A on regulation of IgM levels (i.e., KLH-binding IgM NAb, and total IgM concentration), or B cells biology, or both. This study contributes to increased understanding of (genetic) regulation of KLH-binding NAb levels, and total antibody concentrations.

Nitric oxide hinders antibody clearance from the surface of Trypanoplasma borreli and increases susceptibility to complement-mediated lysis.

Trypanoplasma borreli is an extracellular blood parasite of carp belonging to the same Order (Kinetoplastida) as African trypanosomes. These mammalian parasites have developed different strategies to evade the host immune system including antigenic variation, immunosuppression and clearance of surface-bound antibodies. The latter mechanism allows trypanosomes to use their swimming movement to cause surface-bound antibodies to 'sail' and accumulate at the posterior end of the parasite, to be internalized via the flagellar pocket and be degraded. There is no evidence that T. borreli shows antigenic variation, but during the late phases of infection NO-mediated immunosuppression is observed. High levels of nitric oxide (NO) lead to extensive tissue nitration whereas the parasite itself is not affected. Therefore, the induction of NO has thus far been considered a parasite-driven response with immunosuppressive effects. In the present study, we show that the induction of NO, particularly during the early phase of T. borreli infections, should be re-considered an effective part of the host immune response. We show that T. borreli rapidly removes surface-bound IgM. In addition, moderate concentrations of NO, by hindering surface antibody clearance, maintain high the concentrations of membrane-bound IgM, thereby favoring antibody-dependent complement-mediated parasite lysis. We performed a comprehensive quantitative gene expression analysis of in total seven different complement factors involved in all three activation pathways, differentiating between 1 and 4 isoforms for each complement gene. Our gene expression analysis supports an important role for antibody-dependent complement-mediated lysis of T. borreliin vivo. To our knowledge, NO-dependent inhibition of antibody clearance from the surface of kinetoplastid parasites has not been investigated. Our data support a role for NO as an important player in host-parasite interactions, not only as immune suppressor (late response) but also as immune effector (early response) in infections with bloodstream parasites such as T. borreli.

Allelic discrimination, three-dimensional analysis and gene expression of multiple transferrin alleles of common carp (Cyprinus carpio L.).

We cloned and sequenced four different transferrin (Tf) alleles (C, D, F and G) of European common carp (Cyprinus carpio carpio L.) and studied allelic diversity with respect to differences in sequence, constitutive transcription and three-dimensional structure. Most of the disulfide bonds were conserved between human and carp Tf, and modeling confirmed the overall conservation of the three-dimensional structure of carp Tf. While the iron-binding sites in the C-lobe of carp Tf were completely conserved, in the N-lobe the majority of iron-coordinating residues were not conserved. This may have a serious impact on the ability of carp Tf to bind iron with both the C- and N-lobe. In contrast to human Tf, we could not detect potential N-glycosylation sites in carp Tf, which does not seem to be a glycoprotein. Comparison of the cDNA of the four Tf alleles of carp indicated 21 polymorphic sites of which 13 resulted in non-synonymous changes. Allelic diversity did not seem to influence the overall conservation of carp Tf. Neither the iron binding sites nor the receptor binding of carp Tf seemed influenced by allelic diversity. Possibly, interaction with pathogen-associated receptors for Tf could be influenced by allelic diversity. Basal gene expression of Tf alleles D and G was especially high in carp liver. Although we could detect a higher transcription level of allele D than of Tf allele G in head kidney, thymus and spleen, the differences seem minor with respect to the very high transcription level in liver. Preliminary results with Tf-typed serum suggest a difference in the ability of Tf alleles D and G to modulate LPS-induced NO production in carp macrophages.

Molecular cloning and expression of a Toll receptor in the giant tiger shrimp, Penaeus monodon.

Invertebrates rely completely for their protection against pathogens on the innate immune system. This non-self-recognition is activated by microbial cell wall components with unique conserved molecular patterns. Pathogen-associated molecular patterns (PAMPs) are recognised by pattern recognition receptors (PRRs). Toll and its mammalian homologs Toll-like receptors are cell-surface receptors acting as PRRs and involved in the signalling pathway implicated in their immune response. Here we describe a novel partial Toll receptor gene cloned from a gill library of the giant tiger shrimp, Penaeus monodon, using primers based on the highly conserved Toll/IL-1R (TIR) domain. The deduced amino acid sequence of the P. monodon Toll (PmToll) shows 59% similarity to a Toll-related protein of Apis mellifera. Analysis of the LRRs of shrimp Toll contained no obvious PAMP-binding insertions. Phylogenetic analysis with the insect Toll family shows clustering with Toll1 and Toll5 gene products, and it is less related to Toll3 and Toll4. Furthermore, RT-qPCR shows that PmToll is constitutively expressed in gut, gill and hepatopancreas. Challenge with white spot syndrome virus (WSSV) shows equal levels of expression in these organs. A role in the defence mechanism is discussed. In conclusion, shrimp possess at least one Toll receptor that might be involved in immune defence.

Haemocyte reactions in WSSV immersion infected Penaeus monodon.

White spot syndrome virus (WSSV) has been a major cause of shrimp mortality in aquaculture worldwide in the past decades. In this study, WSSV infection (by immersion) and behaviour recruitment of haemocytes is investigated in gills and midgut, using an antiserum against the viral protein VP28 and a monoclonal antibody recognising haemocytes (WSH8) in a double immunohistochemical staining and in addition transmission electron microscopy was applied. More WSH 8(+) haemocytes were detected at 48 and 72 h post-infection in the gills of infected shrimp compared to uninfected animals. Haemocytes in the gills and midgut were not associated with VP28-immunoreactivity. In the gills many other cells showed virus replication in their nuclei, while infected nuclei in the gut cells were rare. Nevertheless, the epithelial cells in the midgut showed a clear uptake of VP28 and accumulation in supranuclear vacuoles (SNV) at 8h post-infection. However, epithelial nuclei were never VP28-immunoreactive and electron microscopy study suggests degradation of viral-like particles in the SNV. In contrast to the gills, the midgut connective tissue shows a clear increase in degranulation of haemocytes, resulting in the appearance of WSH8-immunoreactive thread-like material at 48 and 72 h post-infection. These results indicate recruitment of haemocytes upon immersion infection in the gills and degranulation of haemocytes in less infected organs, like the midgut.