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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., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

In and around poultry farms, high concentrations of endotoxins are found that have a negative impact on the health of farmers and local residents. However, little is known about the effects of chronic exposure to endotoxins on the health of poultry. The aim of this study was to identify effects of chronic exposure to airborne endotoxins (E. coli LPS) on the immune system, respiratory tract, disease susceptibility and welfare of broilers. Effects of high (HE) and low endotoxin (LE) concentrations on natural antibody titers (NAb), performance and behavior of broilers were determined. After treatment with a respiratory virus infection, infectious bronchitis virus (IBV), mRNA expression of cytokines and Toll-like receptor (TLR) 4 in the lung, tracheal ciliary activity and lesions in the respiratory tract were determined. Endotoxin affected the immune system and respiratory tract, where HE broilers tended to have lower IgM NAb binding Phosphorylcholine-conjugated to Bovine Serum Albumin, and higher interferon (IFN)-α mRNA expression and more lesions in the nasal tissue compared to LE broilers. Furthermore, HE broilers had higher TLR4 mRNA expression compared to LE broilers. However, endotoxin did not affect NAb levels binding Keyhole Limpet Hemocyanin, IFN-β and interleukin-10 mRNA expression, IBV replication or lesions in the lung and trachea. HE and LE broilers further had similar body weight, but HE broilers showed numerically more passive behavior compared to LE broilers. In conclusion, chronic exposure to high airborne endotoxin concentrations affects components of the immune system and respiratory tract in broilers and could therefore influence disease susceptibility., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (© 2022. The Author(s).)

The chicken MHCY region contains members of several gene families including a family of highly polymorphic MHC class I genes that are structurally distinct from their classical class I gene counterparts. Genetic variability at MHCY could impart variability in immune responses, but robust tests for whether or not this occurs have been lacking. Here we defined the MHCY genotypes present in 2 sets of chicken lines selected for high or low antibody response, the Virginia Tech (VT) HAS and LAS, and the Wageningen University (WU) HA and LA lines. Both sets were developed under long-term bidirectional selection for differences in antibody responses following immunization with the experimental antigen sheep red blood cells. Lines in which selection was relaxed (VT HAR and LAR) or lacking (WU C) provided controls. We looked for evidence of association between MHCY genotypes and antibody titers. Chickens were typed for MHCY using a recently developed method based on a multilocus short tandem repeat sequence found across MHCY haplotypes. Five MHCY haplotypes were found segregating in the VT HAS and LAS lines. One haplotype was present only in HAS chickens, and another was present only in LAS chickens with distribution of the remaining 3 haplotypes differing significantly between the lines. In the WU HA and LA lines, there was a similar MHCY asymmetry. The control populations lacked similar asymmetries. These observations support the likelihood of MHCY genetics affecting heritable antibody responses and provide a basis for further investigations into the role of MHCY region genes in guiding immune responses in chickens., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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 β-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 β-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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Verwoolde, Arts, Jansen, Parmentier and Lammers.)

A large variety of clinical manifestation in individual pigs occurs after infection with pathogens involved in porcine respiratory disease complex (PRDC). Some pigs are less prone to develop respiratory disease symptoms. The variation in clinical impact after infection and the recovery capacity of an individual animal are measures of its resilience. In this paper, we examined which ones of a range of animal-based factors (rectal temperature, body weight, skin lesion scores, behavior, natural antibody serum levels, serum levels of white blood cells, and type of T and granulocyte subsets) when measured prior to infection are related to disease severity. These animal-based factors and the interaction with housing regimen of the piglets (conventional or enriched) were modeled using linear regression to predict disease severity using a dataset acquired from a previous study using a well-established experimental coinfection model of porcine reproductive and respiratory syndrome virus (PRRSV) and Actinobacillus pleuropneumoniae . Both PRRSV and A. pleuropneumoniae are often involved in PRDC. Histological lung lesion score of each animal was used as a measure for PRDC severity after infection. Prior to infection, higher serum levels of lymphocytes (CD3 + ), naïve T helper (CD3 + CD4 + CD8 - ), CD8 + (as well as higher relative levels of CD8 + ), and memory T helper (CD3 + CD4 + CD8 + ) cells and higher relative levels of granulocytes (CD172 a ) were related to reduced disease severity in both housing systems. Raised serum concentrations of natural IgM antibodies binding to keyhole limpet hemocyanin (KLH) were also related to reduced disease severity after infection. Increased levels of skin lesions at the central body part (after weaning and before infection) were related to increased disease severity in conventional housing systems only. High resisters showed a lower histological lung lesion score, which appeared unrelated to sex. Body temperature, behavior, and growth prior to infections were influenced by housing regimen but could not explain the variation in lung lesion scores after infection. Raised basal lymphocyte counts and lower skin lesion scores are related to reduced disease severity independent of or dependent on housing system, respectively. In conclusion, our study identifies intrinsic animal-based measures using linear regression analysis that predicts resilience to infections in pigs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 van Dixhoorn, te Beest, Bolhuis, Parmentier, Kemp, van Mourik, Stockhofe-Zurwieden, van Reenen and Rebel.)

Immune maturation of broiler chickens may be affected by management, such as early life feeding strategy (early versus delayed nutrition) or by low or high sanitary conditions (LSC versus HSC). We compared systemic maternal (MAb), natural (NAb), natural auto- (NAAb), and antigen specific antibody (SpAb) levels (IgM, IgY) between broilers (n = 48 per treatment) that received early (EN) or delayed nutrition for 72 h (DN) housed in either low (LSC) or high sanitary conditions (HSC) between 7 and 35 d of age. We found minimal interactions between feeding strategy and sanitary conditions. At 7 d of age, broilers receiving EN compared with DN, had elevated levels of IgM binding keyhole limpet hemocyanin (KLH), phosphoryl-conjugated ovalbumin (PC-OVA), and muramyl dipeptide (MDP), whereas effects of feeding strategy diminished at later ages. In LSC compared with HSC broilers, levels of NAb agglutinating RRBC and sheep red blood cells (SRBC) were already elevated from 14 d of age onwards. At 33 d of age, antibody levels (NAb, NAAb, anti-LPS, anti-MDP) were all elevated in LSC, compared with HSC broilers, for both IgM and IgY, but not IgM against KLH. Western blotting revealed different binding patterns of NAAb against chicken liver homogenate, which may indicate that the NAAb repertoire is affected by antigenic pressure. Our data suggest that antibody levels are affected for an important part by environmental conditions (feeding strategy and sanitary conditions), but minimally by their interaction. However, it remains to be further studied whether the enhanced levels of antibodies as initiated by EN and LSC contribute to enhanced resistance to infectious diseases., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Greater antigenic exposure might accelerate activation and maturation of the humoral immune system. After hatch, commercial broiler chickens can have early (EN) or delayed (DN) access to nutrition, up to 72 h after hatch. The immune system of EN versus DN broilers is likely more exposed to antigens after hatch. This might contribute to activation and maturation of the immune system, but might also influence the development of oral tolerance, thereby altering later life antibody responses. We studied antibody (IgM, IgY, IgA) responses between 21 and 42 d of age in fast-growing EN and DN broilers, kept under low (LSC) or high sanitary conditions (HSC). In a first experiment ( n = 51 broilers), we tested whether early oral exposure to bovine serum albumin (BSA) affected later life antibody responses towards BSA and a novel antigen-rabbit γ-globulin (RGG), under HSC. In a second experiment, a total of 480 EN and DN broilers were housed under either LSC or HSC, and we studied antibody responses against both BSA and RGG ( n = 48 broilers per treatment) and growth performance. Broilers kept under LSC versus HSC, had higher antibody levels and their growth performance was severely depressed. Interactions between feeding strategy (EN versus DN) and sanitary conditions, or main effects of feeding strategy, on natural and specific antibody levels, and growth performance were not observed. Levels of IgA were elevated in EN versus DN broilers, in experiment I and in batch 2 of experiment II, but not in the other batches of experiment II. We concluded that EN versus DN contributes minimally to the regulation of antibody responses, irrespective of antigenic pressure in the rearing environment.

Natural antibodies (NAb) are defined as germline encoded immunoglobulins found in individuals without (known) prior antigenic experience. NAb bind exogenous (e.g., bacterial) and self-components and have been found in every vertebrate species tested. NAb likely act as a first-line immune defense against infections. A large part of NAb, so called natural autoantibodies (NAAb) bind to and clear (self) neo-epitopes, apoptotic, and necrotic cells. Such self-binding antibodies cannot, however, be considered as pathogenic autoantibodies in the classical sense. IgM and IgG NAb and NAAb and their implications in health and disease are relatively well-described in humans and mice. NAb are present in veterinary (and wildlife) species, but their relation with diseases and disorders in veterinary species are much less known. Also, there is little known of IgA NAb. IgA is the most abundant immunoglobulin with essential pro-inflammatory and homeostatic properties urging for more research on the importance of IgA NAb. Since NAb in humans were indicated to fulfill important functions in health and disease, their role in health of veterinary species should be investigated more often. Furthermore, it is unknown whether levels of NAb-isotypes and/or idiotypes can and should be modulated. Veterinary species as models of choice fill in a niche between mice and (non-human) primates, and the study of NAb in veterinary species may provide valuable new insights that will likely improve health management. Below, examples of the involvement of NAb in several diseases in mostly humans are shown. Possibilities of intravenous immunoglobulin administration, targeted immunotherapy, immunization, diet, and genetic modulation are discussed, all of which could be well-studied using animal models. Arguments are given why veterinary immunology should obtain inspiration from human studies and why human immunology would benefit from veterinary models. Within the One Health concept, findings from veterinary (and wildlife) studies can be related to human studies and vice versa so that both fields will mutually benefit. This will lead to a better understanding of NAb: their origin, activation mechanisms, and their implications in health and disease, and will lead to novel health management strategies for both human and veterinary species., (Copyright © 2020 Reyneveld, Savelkoul and Parmentier.)

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., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Barcodes of natural auto-antibody (NAAb) profiles based on staining intensity of isotypes binding numbers of self-(tissue) antigen fragments were suggested as parameters for immune diversity, and related to genetic background and health status in man, rodents and poultry. Here, hens, eggs and hatchlings from chicken lines divergently selected and bred for high (H line) or low (L line) total natural antibodies (NAb) levels in plasma binding keyhole limpet hemocyanin (KLH) at 16 weeks of age were tested for their NAAb repertoire binding chicken liver homogenate (CLH) fragments using quantitative Western immunoblotting. The aims of this study were 1. to detect line differences between the H and L line adult hens, eggs and hatchlings for the IgM and IgG isotypes binding CLH fragments, 2. study the presence of NAAb of both isotypes in yolk and albumen, as well as in hatchlings to detect a maternal NAAb transfer route via the egg to the hatchling, and 3. study whether new self-antigen binding isotypes and idiotypes are present in the hatchling. NAAb binding CLH fragments were found in plasma of adult hens (both IgM and IgG), in yolk (IgG only), and hatchlings (mostly IgG, but low levels of IgM). Auto-profiles of IgM showed homogeneity, while IgG profiles were heterogenic between individual hens and individual hatchlings. Significant higher levels as indicated by staining intensity and number of stained CLH fragments were found in plasma of hens genetically selected for high levels of NAb binding KLH. Lines could be clustered based on their auto-profiles indicating that profiles of self-binding IgM and IgG antibodies are genetically based. Visual comparison, clustering and correlation of hens and their hatchlings showed similarities for the IgG, but not the IgM isotype, indicating maternal transfer of IgG NAAb via the yolk. The IgM profile in the hatchlings on the other hand might represent neonatal self-binding antibody formation. As a consequence, hatchlings initially depend for self-binding antibodies on maternal IgG provision during early life., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 IgG 1 , 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 IgG 1 , 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., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Exposure of yolk androgens can positively stimulate chick growth and competitive ability, but may negatively affect immunity. It has been hypothesized that only chicks from immunologically superior fathers can bear the cost of prenatal exposure to high androgen levels. To test this hypothesis, we paired roosters from two selection lines, one up- and one down-selected for natural antibodies (NAbs), with hens from a control line. We measured yolk testosterone and androstenedione levels, and we injected the treatment group of eggs of each female with testosterone suspended in sesame oil and the control group with sesame oil only. We then measured hatching success and growth, and characterized the humoral and cellular immune responses using three different challenges: a phyto-hemagglutinin, a lipopolysaccharide and a sheep red blood cell challenge. We found that the hatching success, body mass, initial levels of natural antibodies and the chicks' immunological responses to the three different challenges and development were affected neither by paternal immunocompetence nor by treatment. These results do not support the hypothesis that chicks from low-NAb line fathers are more sensitive to testosterone exposure during embryonic development than chicks from high-NAb line fathers., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

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., (© The Author 2019. Published by Oxford University Press on behalf of Poultry Science Association.)

Earlier studies indicated that the inflammatory status of dairy cows in early lactation could not be fully explained by the negative energy balance (NEB) at that moment. The objective of the present study was to determine relationships between inflammatory biomarkers and oxidative stress with uterine health in dairy cows after different dry period lengths. Holstein-Friesian dairy cows were assigned to one of three dry period lengths (0-, 30-, or 60-d) and one of two early lactation rations (glucogenic or lipogenic ration). Cows were fed either a glucogenic or lipogenic ration from 10-d before the expected calving date. Part of the cows which were planned for a 0-d dry period dried themselves off and were attributed to a new group (0 → 30-d dry period), which resulted in total in four dry period groups. Blood was collected ( N = 110 cows) in weeks -3, -2, -1, 1, 2, and 4 relative to calving to determine biomarkers for inflammation, liver function, and oxidative stress. Uterine health status (UHS) was monitored by scoring vaginal discharge (VD) based on a 4-point scoring system (0, 1, 2, or 3) in weeks 2 and 3 after calving. Cows were classified as having a healthy uterine environment (HU, VD score = 0 or 1 in both weeks 2 and 3), nonrecovering uterine environment (NRU, VD score = 2 or 3 in week 3), or a recovering uterine environment (RU, VD score = 2 or 3 in week 2 and VD score= 0 or 1 in week 3). Independent of dry period length, cows with NRU had higher plasma haptoglobin ( P = 0.05) and lower paraoxonase levels ( P < 0.01) in the first 4 weeks after calving and lower liver functionality index ( P < 0.01) compared with cows with HU. Cows with NRU had lower plasma albumin ( P = 0.02) and creatinine ( P = 0.02) compared with cows with a RU, but not compared with cows with HU. Independent of UHS, cows with a 0 → 30-d dry period had higher bilirubin levels compared with cows with 0-, 30-, or 60-d dry period ( P < 0.01). Cows with RU and fed a lipogenic ration had higher levels of albumin in plasma compared with cows with NRU and fed a lipogenic ration ( P < 0.01). In conclusion, uterine health was related to biomarkers for inflammation (haptoglobin and albumin) and paraoxonase in dairy cows in early lactation. Cows which were planned for a 0-d dry period, but dried themselves off (0 → 30-d dry period group) had higher bilirubin levels, which was possibly related to a more severe NEB in these cows. Inflammatory biomarkers in dairy cows in early lactation were related to uterine health in this period., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Keyhole limpet hemocyanin (KLH)-binding natural antibody (NAb) titers in chickens are heritable, and higher levels have previously been associated with a higher survival. This suggests that selective breeding for higher NAb levels might increase survival by means of improved general disease resistance. Chickens were divergently selected and bred for total NAb levels binding KLH at 16 weeks of age for six generations, resulting in a High NAb selection line and a Low NAb selection line. To for test differences in disease resistance, chickens were challenged with avian pathogenic Escherichia coli (APEC) in two separate experiments. Chickens at 8 days of age received one of four intratracheal inoculations of 0.2 mL phosphate buffered saline (PBS): 1) mock inoculate, 2) with 0.2 mL PBS containing 10 8.20  colony-forming units (CFU)/mL APEC, 3) with 0.2 mL PBS containing 10 6.64  CFU/mL APEC, and 4) with 0.2 mL PBS containing 10 7.55  CFU/mL APEC. Mortality was recorded during 7 days post inoculation. Overall, 50-60% reduced mortality was observed in the High line compared to the Low line for all APEC doses. In addition, morbidity was determined of the surviving chickens at 15 days of age. The High line had lower morbidity scores compared to the Low line. We conclude that selective breeding for high KLH-binding NAb levels at 16 weeks of age increase APEC resistance in early life. This study and previous studies support the hypothesis that KLH-binding NAb might be used as an indicator trait for to selective breed for general disease resistance in an antigen non-specific fashion., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

NAb are defined as antigen binding antibodies present without a known previous exposure to this antigen. NAb are suggested to enhance specific antibody (SpAb) responses, but consequences of different NAb levels on immunization are largely unknown. Layer chickens were divergently selected and bred for keyhole limpet hemocyanin (KLH)-binding NAb titers, resulting in a High line and a Low line. In this study, we investigated: (1) the relation of NAb levels with SpAb titers; and (2) the effect of immunization on NAb titers. The 50 highest females of the High line and the 50 lowest females of the Low line of generation 2 were intramuscularly immunized at 33 weeks of age with 1 mL phosphate buffered saline (PBS) containing one of four treatments: (1) negative control (no antigen), (2) 500 µg KLH, (3) 100 µg avian tuberculin purified protein derivative of Mycobacterium avium (PPD), or (4) 250 µg human serum albumin (HuSA). IgM and IgG titers of NAb and SpAb in plasma were determined prior to immunization and weekly for 5 weeks post immunization by indirect ELISA. In addition, antibody affinity was investigated. No differences in SpAb and NAb response against KLH and PPD were observed as a consequence of different NAb titers, but increased and prolonged SpAb and NAb titer responses against HuSA were observed for the High line compared to the Low line. Different natural antibody titers did not impair SpAb dynamics and SpAb affinity. NAb titers were not, or for only short-term, affected by immunization. We show here that NAb may enhance SpAb responses, but that this effect is antigen-dependent. We hypothesize that NAb play a role in general disease resistance through enhancement of the humoral adaptive immune response., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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.

Housing of pigs in barren, stimulus-poor housing conditions may influence their immune status, including antibody responses to (auto-)antigens, and thus affect immune protection, which will influence the onset and outcome of infection. In the present study, we investigated the effects of environmental enrichment versus barren housing on the level of natural (auto-)antibodies (NA(A)b) and their isotypes (IgM and IgG) binding keyhole limpet hemocyanin (KLH), myelin basic protein (MBP), and phosphorycholine conjugated to bovine serum albumin (PC-BSA) in pigs co-infected with porcine reproductive and respiratory syndrome virus (PRRSV) and Actinobacillus pleuropneumoniae (A. pleuropneumoniae). Pigs (n = 56) were housed in either barren or enriched pens from birth to 54 days of age. They were infected with PRRSV on 44 days of age, and with A. pleuropneumoniae 8 days later. Blood samples were taken on 7 different sampling days. Housing significantly affected the overall serum levels of NA(A)b binding KLH, MBP and PC-BSA, and before infection barren housed pigs had significantly higher levels of NA(A)b than enriched housed pigs, except for KLH-IgM and PC-BSA-IgG. Infection only affected the IgM, but not the IgG isotype. Moreover, changes in MBP-IgM and PC-BSA-IgM following infection were different for enriched and barren housed pigs. These results suggest that the effect of infection on NA(A)b is influenced by housing conditions and that NA(A)b, especially IgM may be affected by infection.

Pigs living in commercial husbandry systems may experience both acute stress due to standard management procedures and chronic stress through limitations in their barren housing environment. This might influence their immune status, including antibody responses to neural and danger autoantigens. Levels of natural autoantibody (NAAb)-binding phosphorylcholine-conjugated bovine serum albumin (PC-BSA) and myelin basic protein (MBP) were measured over time in pigs that were kept in environmental enriched v. barren housing, and that underwent a regrouping test. In total, 480 pigs were housed in 80 pens in either barren or straw-enriched pens from 4 through 23 weeks of age. Blood samples were taken from pigs before (week 8), and 3 days after a 24 h regrouping test (week 9), and at 22 weeks of age. Phosphorylcholine-conjugated bovine serum albumin (PC-BSA) and MBP antibody titres in serum were measured using ELISA. Enriched-housed pigs had higher levels of IgM-binding MBP, and tended to have higher levels of IgG-binding MBP and IgA-binding PC-BSA than barren-housed pigs. Each NAAb measured in this study was affected by gender and litter. These results suggest that enriched housing conditions, as well as acute regrouping stress, have an influence on levels of serum NAAb-binding danger and neural antigens in pigs.

Auto-antibody profiles binding liver antigens differed between chicken lines divergently selected for specific antibody responses to SRBC, and were affected by ageing suggesting both genetic and environmental effects. Presence and levels of IgM and IgG antibodies binding chicken liver cell lysate (CLL) fragments in plasma at 5 weeks of age from 10 individual full sibs and their parents from 5 H srbc and 5 L srbc line families was studied to reveal genetic relations. Non-genetic maternal effects were studied by comparing auto-antibody profiles of 36 weeks old hens from 2 other unrelated lines with the profiles from their chicks at hatch. IgM and IgG antibodies from parents and progeny from both H srbc and L srbc lines bound CLL fragments. Significant line and generation differences and their interactions were found for both isotypes. Higher staining of CLL fragments was usually found for H srbc line birds. Lines were clustered by auto-antibody profiles, but staining by birds of both lines in both generations was very individual for IgG and IgM. The current data with full sibs therefore not supported a genetic basis for auto-antibody profiles. IgG but not IgM auto-antibody profiles of chicks correlated with maternal auto-antibody profiles. The results suggest that the auto-antibody repertoire of healthy chickens is largely stochastically initiated and may be affected by environmental challenges during ageing, but genetic mechanisms may underlie staining intensity of individual bound CLL fragments. The present results suggest that identification of fragments or profiles to be used at early age for genetic selection for health traits is not feasible yet. Secondly, the IgM profile of neonatal chickens seems non-organised independent of the maternal profile, but the neonatal IgG profile is much more related with the maternal profile. Consequences of these findings for disease susceptibility or breeding for optimal health are discussed., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Negative energy balance in dairy cows in early lactation has been associated with increased inflammation and oxidative stress in these cows. The objective of this study was to evaluate the effects of dry period (DP) length and dietary energy source on inflammatory biomarkers and oxidative stress in dairy cows. Holstein-Friesian dairy cows (60 primiparous and 107 multiparous) were assigned randomly to a 3 × 2 factorial design with 3 DP length (0, 30, or 60 d) and 2 early lactation rations (glucogenic or lipogenic). Cows were fed a glucogenic or lipogenic ration from 10 d before the expected calving date. Blood was collected in wk -3, -2, -1, 1, 2, and 4 relative to calving. Dry period length affected inflammatory biomarkers and oxidative stress, especially in wk 1 and 2 after calving. Cows with a 0-d DP had higher levels of ceruloplasmin, cholesterol, and reactive oxygen metabolites, and they tended to have higher haptoglobin levels compared with cows with a 30- or 60-d DP. Cows with a 0-d DP had a lower plasma paraoxonase and bilirubin in the first 2 wk after calving and a lower liver functionality index compared with cows with a 60-d DP. Cows of parity >3 fed a glucogenic ration had higher cholesterol levels compared with cows of parity >3 fed a lipogenic ration. No interaction between DP length and ration was present for inflammatory biomarkers or oxidative stress variables. Plasma bilirubin levels for cows with a 0-d DP were negatively related to energy balance and metabolic status in these cows. Moreover, occurrence of clinical health problems (fever, mastitis, metritis, and retained placenta) was 41, 27, and 30% for cows with 0-, 30-, and 60-d DP, respectively. High levels of ceruloplasmin, cholesterol, and reactive oxygen metabolites in cows with 0-d DP were related to the occurrence of health problems in these cows. In conclusion, omitting the DP increased levels of ceruloplasmin, cholesterol, and reactive oxygen metabolites, and decreased levels of bilirubin and paraoxonase in plasma, independent of ration, compared with cows with a 60-d DP. These contrasting effects of DP length on inflammatory status could be explained in part by the improved energy balance and occurrence of health problems in these cows, but was not related to increased somatic cell count in cows with a 0-d DP. Cows with a 0-d DP had better energy balance, but also had higher levels of oxidative stress compared with cows with a 60-d DP. Moreover, occurrence of health problems did not differ between cows with different DP lengths., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

In animals establishment of the intestinal microbial ecosystem is influenced by mucosal immune functions. As mucosal immune functions dynamically change during development of juvenile layer chicken, this study focused on dynamics in the ileal microbiota composition in relation to intestinal immune development. In addition, the levels of immunoglobulin (Ig) in serum and amount of bacteria coated with IgA, a hallmark of intestinal immune maturation, were analysed. The composition of the intestinal microbiota transiently changed at the age of 14-42 days compared to the microbiota composition before and after this period. This temporal deviation in microbiota composition was associated to a temporal increase in transcriptional activity of pro-inflammatory cytokine genes. Furthermore, before week two limited amounts of faecal bacteria were bound by IgM and from week two increasing amounts of bacteria were bound by IgA, reaching a maximal level of 70% of IgA-coated bacteria at 6 weeks of age. These data could indicate that prior to achievement of intestinal homeostasis at 6-10 weeks post hatch, activation of inflammatory pathways cause a temporal disturbance of the microbiota composition. This period of imbalance may be essential for adequate immune development and establishment of intestinal homeostasis.

Natural autoantibodies (NAAb) have a role in maintaining physiological homeostasis and prevention of infections, and have been found in mammalian species tested so far. Albeit NAAb levels rise with age, little is known about the origin, function, regulation and initiation of NAAb in young animals. The present study addressed the presence of IgM and IgG NAAb binding glutamate dehydrogenase (GD), carbonic anhydrase (CA), myosin (MYO) and transferrin (TRANS) from before drinking colostrum until the first 12 weeks of life in plasma of female calves. In addition, NAAb to these four self-antigens were also measured in colostrum and in plasma of their mothers during three weeks before calving. Titers of NAAb binding GD, CA, MYO and TRANS were detected in plasma of cows before calving, in colostrum, and in plasma of calves before and after drinking of colostrum. Levels of NAAb in colostrum were positively related with levels of NAAb in plasma of cows. Before colostrum intake, levels of NAAb in plasma of calves were not related with levels of NAAb in plasma of their mother but were influenced by parity of their mother. After colostrum intake, levels of NAAb in plasma of calves in the first week of life were positively related with levels of NAAb in colostrum. Low NAAb levels in colostrum were related with low NAAb in plasma of calves in the first week of life, but after two weeks of life the relation between colostrum and plasma of calves was absent. In conclusion, NAAb are already present in the unborn calf, and levels of neonatal NAAb during the early weeks of life are affected by levels of maternal NAAb obtained via colostrum., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In earlier studies, natural antibodies (NAb) were related not only to the energy balance (EB) of dairy cows, but also to somatic cell count (SCC) and clinical mastitis (CM). The first objective of our study was to evaluate the effects of dry period length and dietary energy source on titers of NAb binding keyhole limpet hemocyanin (KLH) and lipopolysaccharide (LPS) in plasma and milk, SCC and CM occurrence in dairy cows in two subsequent lactations. Our second objective was to study the relationship between NAb levels and mammary health. Holstein-Friesian dairy cows (N=167) were randomly assigned to three dry period lengths (0, 30 or 60-d) and two early lactation rations (glucogenic or lipogenic). Treatments were repeated during two subsequent lactations (years 1 and 2). In year 2, 19 cows which were planned to have 0-d dry period dried off naturally and were assigned to an additional group 0→30-d dry period. In year 1, cows with a 0-d dry period had a higher SCC, a higher titer of immunoglobulin G (IgG) binding LPS in plasma, and higher titers of IgG and IgM binding KLH and LPS in milk compared with cows with a 30-d or 60-d dry period. In year 2, cows with a 60-d dry period had a lower SCC than cows with a 30-d and 0→30-d dry periods. In year 2, dry period length did not affect NAb titers in plasma or milk. The CM occurrence was 17 percent in year 1 of the experiment and 25 percent in year 2, and did not differ according to dry period lengths or rations. For both years, an increasing titer of IgG binding LPS in plasma was associated with decreased odds of a high SCC and decreased odds of CM occurrence. Also up to three weeks before the CM occurrence, an increasing titer of IgM binding KLH and LPS in plasma was associated with a decreased odds of CM occurrence. In conclusion, omitting the dry period increased SCC, NAb titers in milk and IgG binding LPS in plasma compared with a short (30-d) or conventional (60-d) dry period. The effects on NAb titers, however, were only present in the first year after omitting the dry period and disappeared after repeated omitting the dry period. Moreover, an increasing titer of IgG binding LPS in plasma was associated with decreased odds of high SCC and CM occurrence., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Natural antibodies (NAbs) are mostly IgM antibodies produced without antigenic stimulation and serve as a first line of defence of the immune system. As both natural and specific antibodies are present in animals, NAbs are studied by determining the IgM response to naïve antigens like keyhole limpet hemocyanin (KLH). In this study, we selected cows based on high and low anti-KLH IgM titers, reflecting high and low NAb titers, and determined if the anti-KLH IgM titers were indicative for the recognition of common microbial structures (lipopolysaccharide, lipoteichoic acid and peptidoglycan) and intact bacteria (Escherichia coli and Salmonella Typhimurium). Sera with high NAbs titers showed more IgM and IgG binding to common microbial structures and S. Typhimurium bacteria than sera with low NAbs titers. The same association was observed for IgM binding to E. coli, but not for IgG binding to E. coli. Antibody-mediated complement killing of E. coli and S. Typhimurium in a newly developed bactericidal test was equal between high and low NAb cows. However, relating the outcome of the bactericidal test to the development of mastitis within one and even four years after sampling showed a significant negative correlation implying cows that were less potent in bacterial killing had a higher chance on developing mastitis. In conclusion, sera with high NAbs titers had more antibodies binding to common microbial structures and intact bacteria. Furthermore, the bactericidal test might provide a useful prognostic tool for the development of mastitis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Levels of natural antibodies (NAb) binding keyhole limpet hemocyanin (KLH) in layers were shown to be heritable and to be potential indicative parameters for survival. A proportion of NAb are directed to self-molecules, or slightly changed self-molecules (neo-epitopes), labeled as natural autoantibodies (NAAb). It is unknown whether the levels of NAAb are heritable and genetically correlated. In this paper, we estimated genetic parameters in plasma of healthy layers for IgM and IgG antibodies binding ovalbumin (OVA), myosin (MYO), cardiolipin (CAR), lysozyme (LYS), and the model antigen keyhole limpet hemocyanin (KLH). A linear animal model was used to estimate (co)variance components, heritabilities, and correlations. The estimates of heritabilities ranged from 0.10 to 0.17 for IgM, and 0.02 to 0.11 for IgG, respectively. For both IgM and IgG, high genetic correlations were observed between levels of NAAb binding autoantigens and NAb binding KLH, except for IgG binding KLH and LYS, for which a low genetic correlation was found. Low to moderate phenotypic correlations were found between NAAb and NAb. In addition, significant maternal environmental effects of 0.03, 0.07, and 0.04 were observed for IgM binding OVA, LYS, and KLH, respectively. Results from this study indicated that NAAb or NAb levels in plasma were heritable and could provide tools to identify the health status of birds., (© 2015 Poultry Science Association Inc.)

Natural antibody (NAb) levels and survival rates were evaluated in 4 breeds of laying hens in Ethiopia: indigenous, improved indigenous, exotic layer, and crossbred. Titers of NAb isotypes IgG and IgM binding keyhole limpet hemocyanin (KLH) in serum were measured at 20, 26, 35, and 45 wk age. Repeated-measure ANOVA showed that IgG and IgM levels vary with time within each breed (P < 0.05). Indigenous chickens had significantly (P < 0.05) higher NAb levels at all ages. The Cox proportional hazard analysis showed increased hazard with increased levels of NAbs in the exotic layers (P < 0.05). However, the reduced hazards with increased levels of NAbs were not significant in the improved indigenous and crossbred chickens. Indigenous chickens showed increased hazard with increasing levels of NAb (P > 0.05). We concluded that not only the NAb levels but also the effect of Nabs on survival vary between indigenous and improved breeds. The results indicate that NAb levels are associated with survival in elite (improved) breeds, but are associated with increased hazard in indigenous chickens., (© The Author 2015. Published by Oxford University Press on behalf of Poultry Science Association.)

Natural antibodies (NAb) are defined as antibodies present in individuals without known antigenic challenge. Levels of NAb binding keyhole limpet hemocyanin (KLH) in chickens were earlier shown to be heritable, and to be associated with survival. Selective breeding may thus provide a strategy to improve natural disease resistance. We phenotyped 3,689 white purebred laying chickens for KLH binding NAb of different isotypes around 16 weeks of age. Heritabilities of 0.12 for the titers of total antibodies (IgT), 0.14 for IgM, 0.10 for IgA, and 0.07 for IgG were estimated. We also estimated high, positive genetic, and moderate to high, positive phenotypic correlations of IgT, IgM, IgA, and IgG, suggesting that selective breeding for NAb can be done on all antibody isotypes simultaneously. In addition, a relatively substantial non-genetic maternal environmental effect of 0.06 was detected for IgM, which may reflect a transgenerational effect. This suggests that not only the genes of the mother, but also the maternal environment affects the immune system of the offspring. Breaking strength and early eggshell whiteness of the mother's eggs were predictive for IgM levels in the offspring, and partly explained the observed maternal environmental effects. The present results confirm that NAb are heritable, however maternal effects should be taken into account.

The objective was to study the effect of dry period length in dairy cows on immunoglobulin content and natural antibodies (NAb) titers in colostrum, growth, and plasma natural and specific antibody titers in plasma of calves. Holstein-Friesian dairy cows (n=167) were randomly assigned to 3 dry period lengths (0, 30, or 60 d). Colostrum production, concentration of colostrum IgG and IgM, and titers of NAb (isotypes IgG and IgM) binding keyhole limpet hemocyanin (KLH) and human serum albumin (HuSA) in colostrum were measured. Female calves were immunized with both KLH and HuSA at wk 6 and 10 of life. Titers of NAb and specific antibody (SpAb) for isotypes IgG, IgM, and total immunoglobulin (IgT) binding KLH or HuSA were determined in plasma of female calves. Primary and secondary antibody responses to KLH or HuSA from wk 6 and 10 were expressed as the increase in antibody titers to wk 10 and 11 of life after primary and secondary challenges, respectively. Pregnancy length for cows with a 0-d dry period was 3d shorter compared with cows with a 30- or 60-d dry period. Birth weight of calves from cows with a 0-d dry period was lower compared with calves from cows with a 30-d dry period. Growth of calves until 12 wk of life was not affected by dry period length. Colostrum production and IgG and IgM concentration in colostrum were lower for cows with a 0-d dry period than a 60-d dry period. Natural IgG and IgM titers binding KLH or HuSA were lower in colostrum from cows with a 0-d dry period compared with cows with a 60-d dry period. Natural antibody titers (IgG, IgM, and IgT) binding KLH or HuSA in plasma were lower during the first 2 wk of life for calves from cows with a 0-d dry period compared with calves from cows with a 30- or 60-d dry period. After primary and secondary immunization of calves with KLH and HuSA, SpAb titers of calves were not affected by dry period length. After secondary immunization, the response of IgG and IgT binding KLH was higher in plasma of calves from cows with a 0-d dry period. The results of this study demonstrate that, although omission of the dry period of dairy cows leads to lower plasma NAb titers in calves during the first 2 wk of life, SpAb titers in calves were not affected and even the secondary antibody responses were enhanced compared with calves from cows with a 30- or 60-d dry period., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: Natural autoantibodies (N(a)ab) were found in every species tested so far, and are likely important in maintaining homeostasis., Objectives: (1) To determine N(a)ab in Bos taurus calves, (2) evaluate effects of diet and age on N(a)ab binding repertoires in calves, and (3) delineate bovine liver cell lysate (BLL) antigens related with variation in rumen score and body weight., Animals and Methods: Effects of age and diet on staining of BLL fragments by IgM and IgG antibodies in serum samples collected at 20 or at 26 weeks of age from bull calves either fed a restricted or ad libitum diet were analyzed using quantitative Western blotting. Correlations between fragments stained and grouping of calves were done by Principal Component Analysis (PCA). Redundancy analysis (RDA) was done to relate rumen score and body weight variation at slaughter at 27 weeks of age with stained BLL fragments., Results: In sera from all calves IgM and IgG antibodies binding BLL antigens were found. Corresponding fragments were stained, but quantitative differences in staining intensities were related to diet and age for both IgM and IgG. PCA revealed that age had a greater influence than diet on BLL fragment staining. RDA suggested that staining by IgM or IgG of specific BLL fragments was related with variation in rumen score and body weight., Conclusions and Clinical Importance: Analyses of N(a)ab in serum could be a potential tool to estimate the health status of cattle, and be used to evaluate effects of husbandry practices.

Natural antibodies (NAb) are an important component of the first line of immune defense. Selective breeding for enhanced NAb levels in chickens may improve general disease resistance. It is unknown what the consequences of selection for NAb will be on the productive performance of laying hens. In this paper we describe the genetic relations between NAb titers binding keyhole limpet hemocyanin at 19 wk age and production traits in a white purebred leghorn chicken line observed in several time periods. A linear animal model was used to estimate (co)variance components, heritabilities, and correlations. Negative genetic correlations were found between egg weight and NAb titers, and between egg breaking strength and NAb titers. Positive genetic correlations were found between the feed conversion ratio (consumed feed/egg mass produced) and NAb titers, and egg production and NAb titers. Negative phenotypic correlations were found between body weight and NAb titers, between egg weight and NAb titers, and between egg breaking strength and NAb titers. Positive phenotypic correlations were found between egg production and NAb titers, and feed conversion ratio and NAb titers. In general, phenotypic correlations were more often significant, but less pronounced than genetic correlations. Other production traits were not found to be significant related to NAb titers. These findings suggest that there is a genetic tradeoff between levels of immunity and some production traits, although the underlying mechanism(s) remain(s) unclear. The results suggest possible consequences for production efficiency as a result of selective breeding for improved general disease resistance by natural antibodies., (© 2015 Poultry Science Association Inc.)

To improve the health status (resilience) of dairy cows, levels of natural antibodies (NAb) might be useful. The objective of the present study was to compare levels and to estimate genetic parameters for NAb measured in milk and plasma samples. Titers of NAb IgM and IgG isotype-binding keyhole limpet hemocyanin of 2,919 cows, in both plasma and milk, were measured using ELISA. Analysis revealed that NAb levels in milk significantly increased with parity, whereas they remained constant in plasma. Moderate positive phenotypic correlations were found between NAb levels in milk and in plasma: 0.18 for IgG and 0.40 for IgM. This indicates that NAb from milk and plasma might reflect different aspects of dairy cow health status. However, high genetic correlations were found for NAb in milk and plasma: 0.81 for IgG and 0.79 for IgM. Heritabilities (SE in parentheses) for NAb measured in plasma [0.15 (0.05) for IgG and 0.25 (0.06) for IgM] were higher than heritabilities of NAb measured in milk [0.08 (0.03) for IgG and 0.23 (0.05) for IgM]. Our results indicate that NAb measured in milk and plasma are heritable and likely have a common genetic background, suggesting that NAb levels measured in milk might be useful for genetic improvement of disease resistance., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The aim of the present study was to investigate whether the immune response of broiler chickens is modulated by including different omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) in the maternal diet. Broiler breeder hens (n = 120 birds per group) were fed one of four diets, differing in the ratios of n-6:n-3 PUFAs and eicosapentaenoic acid (EPA):docosahexaenoic acid (DHA). At 28 weeks of age, the eggs produced were incubated to obtain 720 chicks (n = 180 per group). All broiler chicks were fed a control diet and were vaccinated against Newcastle disease virus (NDV). Blood samples were taken at different time points after immunisation with human serum albumin (HuSA) in Freund's adjuvant to determine the acute phase response, antibody response and cytokine production. Addition of EPA to the maternal diet was associated with greater ovotransferrin concentrations post-immunisation, compared to other groups. Altering the ratios of n-6:n-3 PUFA or EPA:DHA in the maternal diet did not affect the offspring in terms of production of caeruloplasmin, α1-acid glycoprotein, interleukin (IL)-1β, IL-6, IL-12 or tumour necrosis factor (TNF)-α. Dietary manipulation of the maternal diet did not influence the specific antibody response to HuSA or NDV, nor did it alter the levels of natural antibody binding to keyhole limpet haemocyanin in the offspring. Thus, maternal supplementation with n-3 PUFAs played a minor role in perinatal programming of the immune response of broiler chickens., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Specificity, antibody isotype distribution and levels, of natural autoantibodies (NAAb) may be potential informative parameters for immune mediated natural disease resistance, immune modulation, and maintenance of physiological homeostasis. In a previous study we detected IgM and IgG antibodies to liver antigens in plasma from 1 year old chickens. Auto-immune profiles directed towards liver antigens differed between chicken lines divergently selected for specific antibody responses to sheep red blood cells. In the present study we measured the presence and typed levels and antibody isotypes (IgG and IgM) of NAAb binding the 'auto-antigen' complex chicken liver cell lysate (CLL) in plasma samples obtained from chickens at 5 weeks and at 1-year of age, respectively, by quantitative western blotting. Extensive staining patterns of plasma antibodies binding CLL were found for both isotypes and at both ages in all birds. At both ages, IgM and IgG bound similar numbers of CLL antigens, which remained almost constant for IgM, whereas the number of IgG stained bands in time was enhanced. Significant differences of binding patterns of NAAb (stained antigen fragments of CLL and staining intensity) were detected between the three different chicken lines at both ages and between both ages, and lines could be clustered on the basis of their auto-antibody profile. The present results indicate that analysis of the plasma NAAb repertoire of poultry like in mammals could provide a way of distinguishing differences of immune competence (as reflected by the selection criterion of antibody responses) between individuals and lines, and could provide tools to select individual birds for health and other traits. The age-dependency of the auto-immune profile suggest that such profiles may also reflect immune maturation, which should be taken into account when relating an auto-immune profile with other traits., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Because of a ban on the use of beak trimming in some European countries, feather pecking is becoming a substantial problem in the layer industry, both from animal welfare and economic points of view. The feather condition score (FCS) as a measure of feather damage has been shown to be closely related to feather pecking behavior in laying hens housed in groups. To obtain a better understanding of genetic and other biological mechanisms underlying feather pecking behavior, data on FCS of a population of 2,724 female offspring from crossing 50 male W1 and 907 female WB purebred lines were used. The offspring of 25 sires were beak-trimmed, and the offspring of another 25 sires were non-beak-trimmed. Titers of plasma natural antibody (NAb) isotypes IgM and IgG binding keyhole limpet hemocyanin at 24 wk of age were measured. Feather condition was scored at 53 wk of age. In the first part of the present study, we estimated genetic parameters for FCS with 2 variance components models: a traditional linear animal model and a model combining direct and associative genetic effects. In the second part of the present study, a trait-based analysis for FCS was conducted to investigate whether NAb isotype titers can explain variation in FCS among individuals, by fitting a linear mixed model. Though the estimated associative genetic variance was substantial, associative effects for FCS were not statistically significant in both populations (P = 0.09 in beak-trimmed birds, and P = 0.08 in non-beak-trimmed birds). This suggests an insufficient number of records on FCS. Individual's NAb isotypes titers did not show direct effect for FCS of itself, but individual's IgG titers showed a suggestive effect on the FCS of cage mates (associative effect) in beak-trimmed laying hens, which need further confirmation.

Immunoglobulins play an important role in maintenance of mucosal homeostasis in the gut. The antigen binding specificity of these immunoglobulins depends for a large part on the hypervariable CDR3 region. To gain knowledge about isotype-specific development of the CDR3 repertoire we examined CDR3 spectratypes at multiple time points between 4 and 70 days post hatch. In order to identify clonal expansions deviation from the normal distribution (SS) and the average CDR3 length was calculated. IgA-CDR3 regions were studied in more detail by DNA sequence analysis at day 7 and 70 and preferential VH pseudogene usage was estimated. The SS of CDR3 repertoires of the IgM, IgG and IgA isotypes successively increased, but for each isotype this increase was transiently. The length of the CDR3 regions decreased with age for IgM becoming similar to the CDR3 length of IgA at day 70. The IgA- and IgG-CDR3 lengths did not change with age. On average, the CDR3 length of IgA was the shortest. IgA CDR3 sequences were similar between animals aged 7 and 70 days. A limited number of pseudogenes was used, and no differences in pseudogene usage were observed between animals aged 7 and 70 days. Of the identified VH pseudogenes, half of the sequences used VH15, whilst a number of the pseudogenes were not used at all. We conclude that CDR3 spectratype profiles change during aging, whilst at the CDR3-sequence level, variation in VH pseudogene usage for ileal IgA is limited suggesting conservation during ontogeny., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Transgenerational epigenetics is becoming more and more important for understanding the variation of physiological responses of individuals to the environment and the inheritance of these responses based on all mechanisms other than the actual DNA nucleotide sequence. Transgenerational epigenetics is the phenomenon that the information of the environment of (usually) a female animal is translated into memory-like responses preparing the offspring. As a consequence, individuals of the next generation may show different phenotypic traits depending whether their mothers were kept under different environmental conditions. This may result in either positive or negative effects on the next-generation individuals, which is different from individuals from mothers that have been kept in a different environment. Transgenerational epigenetic effects have been proposed and indicated for specific immune (T cell and antibody) responses (especially in mammals, but also in birds) and innate immunity (nonvertebrates), but surprisingly very little is known of transgenerational effects on innate immunity in chickens. Given the short lifespan of the chicken and therefore the likely dependence of chicken on innate immune mechanisms, more attention should be given to this arm of immunity and mechanisms of inheritance including transgenerational effects that can be initiated in the breeder generation. In addition, it is becoming evident that innate immunity also underlies metabolic disorders in broilers. In the current paper, we will argue that although very little is known of transgenerational effects of innate immunity in poultry, more attention should be given to this type of study. We will illustrate examples of transgenerational epigenetics, and finally propose strategies that should reveal the presence of transgenerational epigenetic effects on innate immunity in chickens and strategies to modulate breeder birds such that these effects positively affect innate immunity of broilers. It is suggested that a mismatch between breeder environment and broiler environment may account for unwanted effects of innate immunity in the broiler.

Specificity, antibody isotype distribution and levels of natural antibodies (NAb) may be potential informative parameters for immune mediated natural disease resistance, immune modulation, and maintenance of physiological homeostasis. A large proportion of mammalian NAb have affinity for or are directed against self-antigens; so called natural auto antibodies (N(A)Ab). In the present study we showed the presence and typed levels and isotypes (total immunoglobulins, IgG and IgM) of N(A)Ab in plasma binding the 'auto-antigen' complex chicken liver cell lysate (CLL) of one-year old chickens from different genotype and phenotype backgrounds by ELISA and quantitative Western blotting. Higher levels of N(A)Ab binding CLL were found in plasma from chickens genetically selected for high specific antibody responses. In all birds, extensive staining patterns of plasma antibodies binding CLL were found for all isotypes, with IgG binding the highest number of CLL antigens and also showing the highest variation in staining patterns between individuals. Patterns of IgM antibodies binding CLL appeared to be more similar in all lines. Significant differences of binding patterns of N(A)Ab (antigen fragments of CLL and staining intensity) were detected between the different chicken lines, and lines could be clustered on the basis of their auto-antibody profile. In addition, also individual differences within lines were found. The present results indicate that analysis of the levels and the N(A)Ab repertoire of poultry like in mammals could provide a new way of distinguishing differences of immune competence and immune maturation between individuals, and could provide tools to select birds for health traits, or optimize hygiene and husbandry procedures.

Natural antibodies (NAb) are important humoral components of innate immunity. As the first line of defense, NAb provide protection against infection and support adaptive immunity. An earlier study indicated that serum levels of NAb isotypes IgM and IgG at a young age were predictive for survival in non-beak-trimmed purebred laying hens during the laying period. In the present study, genetic parameters of NAb isotypes were estimated and relationships between survival and NAb isotypes levels in crossbred laying hens were investigated. In total, 1,555 beak-trimmed and 1,169 non-beak-trimmed crossbred laying hens were used. Genetic parameters of IgM and IgG titers binding keyhole limpet hemocyanin at 24 wk of age were estimated with a linear animal model. The heritabilities of NAb isotypes IgG and IgM were 0.21 (SE = 0.04) and 0.26 (SE = 0.04), respectively. The genetic correlation between IgG and IgM isotypes was 0.43 (SE = 0.11). These results indicated that NAb isotype titers were heritable traits in the crossbred laying hens. Both NAb isotypes can be selected for simultaneously because the detected positive genetic correlation (0.43, SE = 0.11) between them is positive. Both row and level of the cage were indicated to be associated environmental factors for NAb isotype titers. Different from an earlier study with purebred hens, survival analysis showed no significant associations of survival with NAb isotype titers in beak-trimmed or non-beak-trimmed crossbred hens. Non-health-related causes of mortality, especially in birds with intact beaks, overruled the anticipated relationships between NAb isotype titers and survival.

In an earlier study, serum levels of natural antibody isotypes IgM- and IgG-binding keyhole limpet hemocyanin were found to be indicative for survival through the laying period of hens and therefore considered as promising traits for future implementation in breeding programs for higher survival of layers. In the present study, we first estimated the genetic parameters for the two isotypes at 20, 40 and 65 weeks of age (IgM20, IgM40 and IgM65; IgG20, IgG40 and IgG65). Pooled genetic parameters were estimated from the total population of 2504 hens from nine purebred layer lines, with line included in the model to account for admixture. Moderate heritabilities (0.14-0.44) indicated that selection for isotype titers is feasible, especially for IgM. Secondly, associations between 1022 SNP markers and the above-mentioned six immunological traits were estimated in 650 genotyped hens from the nine lines. The association study was performed across lines to detect markers that are closer to the QTL and have the same phase of association in the entire population. Forty-three significant associations between SNPs and isotype titers were detected. The SNPs of interleukins IL10 and IL19 were associated with both isotypes; SNPs of tripartite motif containing 33 (TRIM33) and IL6 showed significant association with IgG20 and IgM20 respectively; SNPs of heat shock protein 90kDa alpha (cytosolic), class B member 1 (HSP90AB1) were associated with IgG titers at older ages. Some detected SNPs were also reported associated with other immune and behavioral traits., (© 2012 The Authors, Animal Genetics © 2012 Stichting International Foundation for Animal Genetics.)

Environmental particles enter the chicken via several routes. Entry via the respiratory and cloacal routes likely activates immune responses. We studied the localization of simultaneous intratracheally and cloacally applied beads of 2 sizes in the chicken body in time, and when possible, semiquantified the amount of beads. Ten broiler hens, 3.5 wk of age, received 1.25 × 10(9) 1.0-µm beads and 1.05 × 10(7) 10-µm fluorescein isothiocyanate (green) labeled cloacally, and simultaneously the same number and same sizes of tetramethylrhodamine isothiocyanate (red) labeled beads intratracheally. The bursa of Fabricius, lung, liver, kidney, gallbladder, spleen, thymus, small intestine (upper ileum), cecum, intestinal luminal contents, aerated bones, feces, and blood, from 2 chickens per moment were sampled at 1 h, 6 h, 24 h, 48 h, and 1 wk after challenge and studied for the presence of beads using fluorescence microscopy. The highest amount of beads was found in organs closest to the application site after 1 h (i.e., the lungs for red beads, and the bursa for green beads). All tissue samples showed all 4 types of beads at all time moments, most of them within 1 h. Lower levels of beads were found in lungs and bursa after 6 h and in all other organs after 24 h, except for the kidneys where levels declined after 48 h. Surprisingly, beads were found in thymus tissue and only relatively few beads were found in the spleen. At 1 h, 1-µm intratracheally applied red beads were also found in the cecal luminal content and cecal tissue, but not in the small intestinal luminal content, suggesting that ceca are capable of excreting small particles entering the body via the respiratory route. The presence of nondegradable and nonimmunogenic beads of different sizes in all sampled organs throughout the whole chicken body for 7 d suggested potentially negative chronic health and welfare risks for the chicken of environmental particles.

Lipopolysaccharides (LPS) are present in high levels in the air of chicken houses and likely have immune-modulating effects. In this study, layers from the 30th generation of a divergent selection experiment for humoral immune reactivity to subcutaneously administered sheep red blood cells were concurrently intratracheally challenged with human serum albumin (HuSA) and LPS at 7 and 12 wk of age following a crossing over design. Chickens selected for high humoral immune reactivity (H line) showed higher specific antibody responses to intratracheally administered HuSA and a higher level of natural antibody binding keyhole limpet hemocyanin compared with chickens genetically selected for low humoral immune reactivity (L line), and were also more sensitive to immune modulation by LPS at 7 wk of age. Body weight gain was negatively affected by LPS at 7 wk of age in the L line, but after 12 wk of age in the H line. Egg production was lower and delayed in the H line. We conclude that these chicken selection lines differ in sensitivity to the environment (LPS), and consequently may therefore also react differently to infection, vaccinations, and other immune responses. In addition, selection for immune responsiveness affected growth and egg lay.

Positive effects of yeast concentrate on immunity and performance of weaned piglets have been reported. However, the effects on innate immunity were not examined. Natural antibodies (NAb) are part of innate immunity and have been related to health and survival in fish, poultry, rodents, and man. Yeast cell walls may also affect innate immunity of weaned piglets. We studied the effect of Nuklospray ProHealth containing a spray dried blend of dairy-based feed ingredients, vegetable fats, and processed yeast cell walls as protein source on NAb levels and performance of weaned piglets. A total of 120 piglets weaned at 28 d of age were assigned 2 treatments comprising a control diet and an experimental diet with the test product. Piglets were housed in groups of 6 during the first 4 weeks after weaning. Blood samples of 20 healthy nonmedicated piglets per treatment were taken at days 0, 14, and 28 after weaning and analyzed for NAb levels binding keyhole limpet hemocyanin by an indirect ELISA procedure. Performance parameters also were determined. Overall, the experimental diet tended to improve feed intake (574 vs. 522 g/d; P < 0.1), ADG (449 vs. 412 g/d; P < 0.1), and final BW (21.4 vs. 20.3 kg; P = 0.08) compared to the control diet. No differences were found in feed conversion ratio or fecal score. At day 0, no differences in NAb levels were found, but on day 14 after weaning, NAb levels of piglets fed the experimental diet were significantly higher than of piglets fed the control diet (2.05 vs. 1.70; P < 0.05). On day 28 after weaning no differences were found. These results indicate that day 14 postweaning levels of NAb as a parameter of innate immunity were improved and indicate a tendency for improvement of postweaning performance of piglets fed diets supplemented with Nuklospray ProHealth.

We studied the effects of a concurrent challenge on slow-growing broilers with 1) airborne particles of 2 sizes: fine dust (smaller than 2.5 microns) and coarse dust (between 2.5 and 10 microns) that were directly collected from a broiler house and 2) lipopolysaccharide on intratracheal immunizations with the specific antigen human serum albumin (HuSA) and measured primary and secondary systemic (total) antibody responses and (isotype-specific) IgM, IgG, and IgA responses at 3 and 7 wk of age. All treatments affected immune responses at several ages, heart morphology, and BW gain, albeit the latter only temporarily. Dust particles significantly decreased primary antibody (IgT and IgG) responses to HuSA at 3 wk of age but enhanced IgM responses to HuSA at 7 wk of age. Dust particles decreased secondary antibody responses to HuSA, albeit not significantly. All of the birds that were challenged with dust particles showed decreased BW gain after the primary but not after the secondary challenge. Relative heart weight was significantly decreased in birds challenged with coarse dust, fine dust, lipopolysaccharide, and HuSA at 3 wk of age, but not in birds challenged at 7 wk of age. Morphology (weight, width, and length) of hearts were also affected by the dust challenge at 3 wk of age. The present results indicate that airborne dust particles obtained from a broiler house when intratracheally administered at an early age affect specific humoral immune responsiveness and BW gain of broilers to simultaneously administered antigens differently than when administered at a later age. The hygienic status of broiler houses at a young age may be of importance for growth and immune responsiveness, and consequently, for vaccine efficacy and disease resistance in broilers. The consequences of our findings are discussed.

Natural antibodies (NAb) are defined as antibodies that circulate in normal healthy individuals under the absence of deliberate antigenic stimulation. Two types of NAb are distinguished: NAb towards exogenous antigens and NAb towards autoantigens (N(A)Ab). The objectives of the current study were threefold. First, we studied the relation between metabolic health and concentrations of NAb binding keyhole limpet hemocyanin (KLH) or lipopolysaccharide (LPS) in milk and plasma of dairy cows in early lactation. Second, we determined the presence of N(A)Ab binding transferrin, myosin and thyroglobulin in bovine milk. Third, we studied the relation between N(A)Ab in bovine milk and mammary health. For the first objective, dairy cows were either fed a control (C) (n=8) or a diet where 2 kg of concentrates were replaced by an iso-energetic concentrate containing marine algae (ALG) from week -3 till 8 postpartum (experiment 1). Plasma and milk samples were analyzed weekly for NAb binding either KLH or LPS. Plasma was analyzed for glucose, non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHBA). For the second and third objective, milk samples were collected weekly from 96 dairy cows from week 2 till 9 postpartum and analyzed for milk composition and N(A)Ab binding myosin, transferrin and thyroglobulin (experiment 2). For both datasets, N(A)Ab titers are expressed as (2)log values of the highest dilution giving a positive reaction. Data are expressed as means ± SEM. Repeated observations were analyzed in a mixed model. In experiment 1, no diet effect (P>0.05) was observed on NAb binding LPS in plasma or milk, NAb binding KLH in milk was greater (P=0.05) for cows fed the control diet. Concentration of NAb binding KLH and LPS in plasma was negatively related to plasma NEFA concentration (P<0.05). In experiment 2, NAb binding myosin (5.66 ± 0.06), thyroglobulin (4.85 ± 0.06), and transferrin (5.76 ± 0.07) were identified in milk. Clinical mastitis incidence (9%) tended to be positively related to concentration of NAb binding myosin (P=0.06) and negatively related to Nab binding transferrin (P=0.08). In conclusion, NAb binding KLH and LPS in plasma and milk are related to metabolic health, as indicated by plasma NEFA concentration. Furthermore, this study demonstrates the presence of N(A)Ab in bovine milk and shows trends for a relation between N(A)Ab binding auto-antigens and mastitis. Future studies should confirm these trends and shed light on the predictive value of N(A)Ab in bovine milk for mammary health., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Innate immunity plays an important role in preventing (barrier function) or combating infection (effector function). An important humoral component of innate immunity is formed by natural antibodies (NAb). The objectives of this study were to determine presence, variation among cows and repeatability within cows over time of total NAb titers directed to the pathogen-associated molecular patterns lipopolysaccharide, lipoteichoic acid (LTA) and peptidoglycan, and titers of NAb directed to the glycoprotein keyhole limpet hemocyanin in milk and plasma of individual cows. Furthermore in milk the antibody isotypes IgG1, IgG2, IgM and IgA binding LTA were analyzed. Ten milk and blood samples were obtained from each of 20 clinically healthy dairy cows from first to seventh parity during a period of 3 weeks. Total NAb binding lipopolysaccharide, LTA, peptidoglycan, and keyhole limpet hemocyanin were detected in milk and plasma, with titers considerably higher in plasma than in milk. Total NAb titers showed significant variation among cows, and repeatability within cows over time (ranging from 0.60 to 0.93). Titers of NAb in milk and plasma were positively correlated (correlation ranging from 0.69 to 0.91). Natural antibodies in milk binding LTA were of all 4 isotypes tested, although IgG2 was on average only present at low titers. All 4 isotypes in milk binding LTA also showed variation among cows, and repeatability within cows over time (ranging from 0.84 to 0.92). We conclude that NAb can be measured in a consistent and repeatable manner in bovine milk and blood plasma., (Copyright © 2011 Elsevier B.V. All rights reserved.)