Low gH/gL (Sub)Species-Specific Antibody Levels Indicate Elephants at Risk of Fatal Elephant Endotheliotropic Herpesvirus Hemorrhagic Disease.

Elephant endotheliotropic herpesviruses (EEHVs), of which eleven (sub)species are currently distinguished, infect either Asian (Elephas maximus) or African elephants (Loxodonta species). While all adult elephants are latently infected with at least one EEHV (sub)species, young elephants, specifically those with low to non-detectable EEHV-specific antibody levels, may develop fatal hemorrhagic disease (EEHV-HD) upon infection. However, animals with high antibody levels against EEHV(1A) gB, an immunodominant antigen recognized by antibodies elicited against multiple (sub)species, may also occasionally succumb to EEHV-HD. To better define which animals are at risk of EEHV-HD, gB and gH/gL ELISAs were developed for each of the Asian elephant EEHV subspecies and assessed using 396 sera from 164 Asian elephants from European zoos. Antibody levels measured against gB of different (sub)species correlated strongly with one another, suggesting high cross-reactivity. Antibody levels against gH/gL of different subspecies were far less correlated and allowed differentiation between these (sub)species. Importantly, while high gB-specific antibody levels were detected in the sera of several EEHV-HD fatalities, all fatalities (n = 23) had low antibody levels against gH/gL of the subspecies causing disease. Overall, our data indicate that (sub)species-specific gH/gL ELISAs can be used to identify animals at risk of EEHV-HD when infected with a particular EEHV (sub)species.

Characterization of polarization states of canine monocyte derived macrophages.

Macrophages can reversibly polarize into multiple functional subsets depending on their micro-environment. Identification and understanding the functionality of these subsets is relevant for the study of immune­related diseases. However, knowledge about canine macrophage polarization is still in its infancy. In this study, we polarized canine monocytes using GM-CSF/IFN- γ and LPS towards M1 macrophages or M-CSF and IL-4 towards M2 macrophages and compared them to undifferentiated monocytes (M0). Polarized M1 and M2 macrophages were thoroughly characterized for morphology, surface marker features, gene profiles and functional properties. Our results showed that canine M1-polarized macrophages obtained a characteristic large, roundish, or amoeboid shape, while M2-polarized macrophages were smaller and adopted an elongated spindle-like morphology. Phenotypically, all macrophage subsets expressed the pan-macrophage markers CD14 and CD11b. M1-polarized macrophages expressed increased levels of CD40, CD80 CD86 and MHC II, while a significant increase in the expression levels of CD206, CD209, and CD163 was observed in M2-polarized macrophages. RNAseq of the three macrophage subsets showed distinct gene expression profiles, which are closely associated with immune responsiveness, cell differentiation and phagocytosis. However, the complexity of the gene expression patterns makes it difficult to assign clear new polarization markers. Functionally, undifferentiated -monocytes, and M1- and M2- like subsets of canine macrophages can all phagocytose latex beads. M2-polarized macrophages exhibited the strongest phagocytic capacity compared to undifferentiated monocytes- and M1-polarized cells. Taken together, this study showed that canine M1 and M2-like macrophages have distinct features largely in parallel to those of well-studied species, such as human, mouse and pig. These findings enable future use of monocyte derived polarized macrophages particularly in studies of immune related diseases in dogs.

Perforin and granzyme A release as novel tool to measure NK cell activation in chickens.

Natural killer (NK) cells are cytotoxic lymphocytes that are present in the circulation but also in many organs including spleen and gut, where they play an important role in the defense against infections. Interaction of NK cells with target cells leads to degranulation, which results in the release of perforin and granzymes in the direct vicinity of the target cell. Chicken NK cells have many characteristics similar to their mammalian counterparts and based on similarities with studies on human NK cells, surface expression of CD107 was always presumed to correlate with granule release. However, proof of this degranulation or in fact the actual presence of perforin (PFN) and granzyme A (GrA) in chicken NK cells and their release upon activation is lacking. Therefore, the purpose of the present study was to determine the presence of perforin and granzyme A in primary chicken NK cells and to measure their release upon degranulation, as an additional tool to study the function of chicken NK cells. Using human specific antibodies against PFN and GrA in fluorescent and confocal microscopy resulted in staining in chicken NK cells. The presence of PFN and GrA was also confirmed by Western blot analyses and its gene expression by PCR. Stimulation of NK cells with the pectin SPE6 followed by flow cytometry resulted in reduced levels of intracellular PFN and GrA, suggesting release of PFN and GrA. Expression of PFN and GrA reversely correlated with increased surface expression of the lysosomal marker CD107. Finally it was shown that the supernatant of activated NK cells, containing the NK cell granule content including PFN and GrA, was able to kill Escherichia coli. This study correlates PFN and GrA release to activation of chicken NK cells and establishes an additional tool to study activity of cytotoxic lymphocytes in chickens.

Cellular and humoral immune responsiveness to inactivated Leptospira interrogans in dogs vaccinated with a tetravalent Leptospira vaccine.

Vaccination is commonly used to protect dogs against leptospirosis, however, memory immune responses induced by canine Leptospira vaccines have not been studied. In the present study, antibody and T cell mediated responses were assessed in dogs before and 2 weeks after annual revaccination with a commercial tetravalent Leptospira vaccine containing serogroups Canicola and Australis. Vaccination significantly increased average log2 IgG titers from 6.50 to 8.41 in year 1, from 5.99 to 7.32 in year 2, from 5.32 to 8.32 in year 3 and from 5.32 to 7.82 in year 4. The CXCL-10 levels, induced by in vitro stimulation of PBMC with Canicola and Australis, respectively, significantly increased from 1039.05 pg/ml and 1037.38 pg/ml before vaccination to 2547.73 pg/ml and 2730.38 pg/ml after vaccination. IFN-γ levels increased from 85.60 pg/ml and 178.13 pg/ml before vaccination to 538.62 pg/ml and 210.97 pg/ml after vaccination. The percentage of proliferating CD4+ T cells in response to respective Leptospira strains significantly increased from 1.43 % and 1.25 % before vaccination to 24.11 % and 14.64 % after vaccination. Similar responses were also found in the CD8+ T cell subset. Vaccination also significantly enhanced the percentages of central memory CD4+ T cells from 12 % to 26.97 % and 27.65 %, central memory CD8+ T cells from 3 % to 9.47 % and 7.55 %, and effector CD8+ T cells from 3 % to 7.6 % and 6.42 %, as defined by the expression of CD45RA and CD62L, following stimulation with Canicola and Australis, respectively. Lastly, enhanced expression of the activation marker CD25 on T cells after vaccination was found. Together, our results show that next to IgG responses, also T cell responses are induced in dogs upon annual revaccination with a tetravalent Leptospira vaccine, potentially contributing to protection.

Transcriptome and proteome analysis of innate immune responses to inactivated Leptospira and bivalent Leptospira vaccines in canine 030-D cells.

Mandatory potency testing of Leptospira vaccine batches relies partially on in vivo procedures, requiring large numbers of laboratory animals. Cell-based assays could replace in vivo tests for vaccine quality control if biomarkers indicative of Leptospira vaccine potency are identified. We investigated innate immune responsiveness induced by inactivated L. interrogans serogroups Canicola and Icterohaemorrhagiae, and two bivalent, non-adjuvanted canine Leptospira vaccines containing the same serogroups. First, the transcriptome and proteome analysis of a canine monocyte/macrophage 030-D cell line stimulated with Leptospira strains, and vaccine B revealed more than 900 DEGs and 23 DEPs in common to these three stimuli. Second, comparison of responses induced by vaccine B and vaccine D revealed a large overlap in DEGs and DEPs as well, suggesting potential to identify biomarkers indicative of Leptospira vaccine quality. Because not many common DEPs were identified, we selected seven molecules from the identified DEGs, associated with pathways related to innate immunity, of which CXCL-10, IL-1ß, SAA, and complement C3 showed increased secretion upon stimulation with both Leptospira vaccines. These molecules could be interesting targets for development of biomarker-based assays for Leptospira vaccine quality control in the future. Additionally, this study contributes to the understanding of the mechanisms by which Leptospira vaccines induce innate immune responses in the dog.

EEHV1A glycoprotein B subunit vaccine elicits humoral and cell-mediated immune responses in mice.

Asian elephants are an endangered species facing many threats, including severe hemorrhagic disease (HD) caused by the elephant endotheliotropic herpesvirus (EEHV). EEHV-HD is the leading cause of death in captive juvenile Asian elephants in North America and Europe, and also affects elephants in their natural range countries. Significant challenges exist for successful treatment of EEHV-HD, which include timely recognition of disease onset and limited availability of highly effective treatment options. To address this problem, our goal is to prevent lethal disease in young elephants by developing a vaccine that elicits robust and durable humoral and cell-mediated immunity against EEHV. EEHV glycoprotein B (gB) is a major target for cellular and humoral immunity in elephants previously exposed to EEHV. Therefore, we generated a vaccine containing recombinant EEHV1A gB together with a liposome formulated TLR-4 and saponin combination adjuvant (SLA-LSQ). CD-1 mice that received one or two vaccinations with the vaccine elicited significant anti-gB antibody and polyfunctional CD4+ and CD8+ T cell responses, while no adverse effects of vaccination were observed. Overall, our findings demonstrate that an adjuvanted gB protein subunit vaccine stimulates robust humoral and cell-mediated immune responses and supports its potential use in elephants.

Young elephants in a large herd maintain high levels of elephant endotheliotropic herpesvirus-specific antibodies and do not succumb to fatal haemorrhagic disease.

Elephant endotheliotropic herpesviruses (EEHVs) have co-existed with elephants for millions of years, yet may cause fatal haemorrhagic disease (EEHV-HD), typically in elephants between 1 and 10 years of age. EEHV is omnipresent in (sub)adult elephants, and young elephants with low EEHV-specific antibody levels are at risk for EEHV-HD, suggesting that fatal disease may occur due to an insufficiently controlled primary infection. To further address this hypothesis, sera of three large elephant cohorts were subjected to a multiple EEHV species ELISA: (I) 96 Asian elephants between 0 and 57 years, including 13 EEHV-HD fatalities, from European zoo herds typically sized five to six elephants, (II) a herd of 64 orphaned elephants aged 0-15 years at the Elephant Transit Home in Sri Lanka and (III) 31 elephants aged 8-63 years, part of a large herd of 93 elephants at Pinnawala Elephant Orphanage, Sri Lanka. All Sri Lankan elephants showed high EEHV-specific antibody levels regardless of their age. While antibody levels of most European zoo elephants were comparable to those of Sri Lankan elephants, the average antibody level of the European juveniles (1-5 years of age) was significantly lower than those of age-matched Sri Lankan individuals. Moreover, the European juveniles showed a gradual decrease between 1 and 4 years of age, to be attributed to waning maternal antibodies. Maintenance of high levels of antibodies in spite of waning maternal antibodies in young Sri Lankan elephants is likely due to the larger herd size that increases the likelihood of contact with EEHV-shedding elephants. Together with the observation that low levels of EEHV-specific antibodies correlate with increased numbers of EEHV-HD fatalities, these results suggest that infection in presence of high maternal antibody levels may protect calves from developing EEHV-HD, while at the same time activating an immune response protective in future encounters with this virus.

Activation of Canine, Mouse and Human TLR2 and TLR4 by Inactivated Leptospira Vaccine Strains.

Canine Leptospira vaccines contain inactivated strains of pathogenic Leptospira, the causative agents of leptospirosis. For an effective response to vaccination, activation of the innate immune system via pattern recognition receptors such as TLRs is crucial. However, it is not known which TLRs are activated by Leptospira in dogs. To investigate the involvement of canine TLR2, TLR4, and TLR5 in the recognition of Leptospira, we stimulated canine moDC and reporter cells expressing canine TLR2 with either whole-inactivated bacteria or purified LPS of Leptospira strains, representing the serogroups generally used in canine leptospirosis vaccines. Using the endotoxin neutralizing reagent polymyxin B and TLR4 antagonist RS-LPS, we demonstrate that Leptospira LPS and canine TLR4 are involved in IL-1ß production as well as in the uptake of inactivated Leptospira in canine moDC. Furthermore, polymyxin B only partially inhibited IL-1ß production induced by inactivated Leptospira, suggesting that next to TLR4, also other TLRs may be involved. The observed activation of canine TLR2-expressing reporter cells by inactivated Leptospira strains indicates that TLR2 could be one of these TLRs. Next, we analyzed TLR2 and TLR4 activating capabilities by the same Leptospira strains using human and mouse TLR-expressing reporter cells. Inactivated Leptospira and leptospiral LPS activated not only mouse, but also human TLR4 and this activation was shown to be LPS dependent in both cases. Additionally, inactivated Leptospira activated mouse and human TLR2-expressing reporter cell lines. In our study, we could not identify significant species differences in the recognition of Leptospira by TLR2 and TLR4 between dog, human and mouse. Lastly, we show that these inactivated Leptospira strains are recognized by both mouse and human TLR5 reporter cells only after exposure to additional heat-treatment. Unfortunately, we were not able to confirm this in the canine system. Our data show that TLR2 and TLR4 are involved in the recognition of Leptospira strains used in the production of canine Leptospira vaccines. This study contributes to the understanding of Leptospira-induced innate immune responses in dogs, humans, and mice. Future studies are needed to further explore the role of canine TLR2, TLR4 and TLR5 in the induction of vaccine-mediated immunity against Leptospira.

Effects of E scherichia coli Nissle 1917 on the Porcine Gut Microbiota, Intestinal Epithelium and Immune System in Early Life.

Early in life and particularly around weaning, piglets are susceptible to infections because of abrupt social, environmental, and dietary changes. Dietary interventions with probiotic bacteria have gained popularity because of the increased awareness of the direct link between diet and health. In this study, piglets received the probiotic strain Escherichia coli Nissle 1917 (EcN) or a control treatment perorally from day 2 after birth until 2 weeks post-weaning. To investigate spatio-temporal effects of EcN on the gut microbiota composition, intestinal epithelial gene expression and immune system, feces, digesta, blood, scraping material and mesenteric lymph node tissue were collected at different time points. In addition, oral vaccinations against Salmonella enterica serovar Typhimurium were administered on days 21 and 45 of the study to assess the immunocompetence. EcN-treated pigs showed a reduced diversity of taxa within the phylum Proteobacteria and a lower relative abundance of taxa within the genus Treponema during the pre-weaning period. Moreover, EcN induced T cell proliferation and Natural Killer cell activation in blood and enhanced IL-10 production in ex vivo stimulated mesenteric lymph node cells, the latter pointing toward a more regulatory or anti-inflammatory state of the local gut-associated immune system. These outcomes were primarily observed pre-weaning. No significant differences were observed between the treatment groups with regards to body weight, epithelial gene expression, and immune response upon vaccination. Differences observed during the post-weaning period between the treatment groups were modest. Overall, this study demonstrates that the pre-weaning period offers a 'window of opportunity' to modulate the porcine gut microbiota and immune system through dietary interventions such as EcN supplementation.

Long-chain glucomannan supplementation modulates immune responsiveness, as well as intestinal microbiota, and impacts infection of broiler chickens with Salmonella enterica serotype Enteritidis.

The zoonotic pathogen Salmonella enterica serotype Enteritidis (SE) causes severe disease in young chickens. Restriction on antibiotic use requires alternative SE control strategies such as nutritional solutions to improve the resistance of chickens. In this study, chickens were fed long-chain glucomannan (GM) or standard diet and challenged with SE at seven days of age. During 21 days post-infection (dpi), we determined numbers and responsiveness of natural killer (NK) and T cells in ileum and spleen, and SE-specific antibody titers in serum. Microbiota compositions in ileum and caeca were determined, as well as correlations of these with numbers and function of immune cells. Some of the samples in the control group had numerically higher CFUs than the GM-treated group. In addition, the relative abundance of SE based on DNA assessment was significantly lower at 21 dpi upon GM supplementation. At 3 dpi, numbers of intraepithelial NK cells were significantly higher, while activation of intraepithelial NK cells (7 dpi), numbers of intraepithelial cytotoxic CD8+ T cells (14 dpi) and SE-specific antibodies (14 dpi) were numerically higher. Furthermore, relative abundance of the commensal lactic acid bacteria (LAB) significantly increased with GM supplementation post-infection. Higher relative abundance of streptococci was associated with reduced SE in ileal and caecal contents at 21 dpi. Relative abundance of streptococci negatively correlated with SE counts and positively correlated with NK cell activation and SE-specific antibodies, which suggests involvement of the commensal LAB in NK cell responsiveness. These results indicate that GM supplementation modulates the immune system, intestinal microbiota and impacts SE infection of young chickens.

Lepra Bubalorum, a Potential Reservoir of Mycobacterium leprae.

In 1926, a mycobacterial skin disease was observed in water buffaloes by researchers in Indonesia. The disease was designated as skin tuberculosis, though it was hypothesized that it might be a form of leprosy or a leprosy-like disease. In a follow-up study (Ph.D. thesis Lobel, 1934, Utrecht University, Netherlands) a similar nodular skin disease was described in Indonesian water buffaloes and named "lepra bubalorum" or "nodular leprosy." Two decades later Kraneveld and Roza (1954) reported that, so far, the diagnosis lepra bubalorum had been made in 146 cases in Indonesia. After a final series of research reports by Indonesian veterinarians in 1961, no subsequent cases were published. Based on information from these reports, it can be concluded that, even though evidence of nerve involvement in buffaloes was not reported, similarities exist between lepra bubalorum and Hansen's disease (leprosy), i.e., nodular skin lesions with a chronic course and microscopically granulomatous reactions with AFB in globi in vacuoles. This raises the question as to whether these historical cases might indeed have been caused by Mycobacterium leprae, Mycobacterium lepromatosis or another representative of the M. leprae complex. The future use of state-of-the-art molecular techniques may answer this question and may also help to answer the question whether water buffaloes should be considered as a potential natural reservoir of the causative pathogen of Hansen's disease.

Prevalence and Demographic Risk Factors of Mycobacterium tuberculosis Infections in Captive Asian Elephants (Elephas maximus) Based on Serological Assays.

To address putative TB statuses of elephants and to identify and quantify potential demographic risk factors for TB, three ELISAs specific for different mycobacterial antigens (ESAT6, CFP10, MPB83) and the TB Stat-Pak assay were used as surrogate serological markers for TB infection in elephants. In view of the low number of animals of which the infected status could be confirmed (4 out of 708) Latent Class Analyses of TB serology test outcomes was used to predict the putative TB status of each of 708 elephants as positive (17.3%), inconclusive (48.7%), or negative (34%) when assessed on a population basis. Correlation between test performance of the individual assays was high between the ELISAs, but low with that of the TB Stat-Pak assay. Risk factors, assessed based on cut off values for each of the ELISAs determined by ROC analysis, included sex, BCS, age, working time, feed type, management system, camp size and region. Old age elephants were more likely to show a positive TB serology test outcome, than younger ones. Elephants working 7 h per day and the ones in good condition BCS (7-11) were less likely to be positive in TB serology testing. In addition, fewer animals in the large camp size (31-50 elephants) were found to be positive in ELISA tests, compared to elephants in the other camp sizes. In this study, the North region had the lowest percentages of elephants with positive TB test outcome, the West region and to a lesser extend the other regions showed clearly higher percentages of positive animals. Even though assays used in the present study have not been validated yet, results obtained showed promise as diagnostic or screening tests. For the diagnosis of animals suspected to be infected, the ELISA tests, once further optimized for the individual antigens, can be used in parallel. For screening of complete camps for presence or absence of infection, a single optimized ELISA test can be utilized.

The Interplay between Salmonella and Intestinal Innate Immune Cells in Chickens.

Salmonellosis is a common infection in poultry, which results in huge economic losses in the poultry industry. At the same time, Salmonella infections are a threat to public health, since contaminated poultry products can lead to zoonotic infections. Antibiotics as feed additives have proven to be an effective prophylactic option to control Salmonella infections, but due to resistance issues in humans and animals, the use of antimicrobials in food animals has been banned in Europe. Hence, there is an urgent need to look for alternative strategies that can protect poultry against Salmonella infections. One such alternative could be to strengthen the innate immune system in young chickens in order to prevent early life infections. This can be achieved by administration of immune modulating molecules that target innate immune cells, for example via feed, or by in-ovo applications. We aimed to review the innate immune system in the chicken intestine; the main site of Salmonella entrance, and its responsiveness to Salmonella infection. Identifying the most important players in the innate immune response in the intestine is a first step in designing targeted approaches for immune modulation.

Overcoming scientific barriers in the transition from in vivo to non-animal batch testing of human and veterinary vaccines.

INTRODUCTION: Before release, vaccine batches are assessed for quality to evaluate whether they meet the product specifications. Vaccine batch tests, in particular of inactivated and toxoid vaccines, still largely rely on in vivo methods. Improved vaccine production processes, ethical concerns, and suboptimal performance of some in vivo tests have led to the development of in vitro alternatives. AREAS COVERED: This review describes the scientific constraints that need to be overcome for replacement of in vivo batch tests, as well as potential solutions. Topics include the critical quality attributes of vaccines that require testing, the use of cell-based assays to mimic aspects of in vivo vaccine-induced immune responses, how difficulties with testing adjuvanted vaccines in vitro can be overcome, the use of altered batches to validate new in vitro test methods, and how cooperation between different stakeholders is key to moving the transition forward. EXPERT OPINION: For safety testing, many in vitro alternatives are already available or at an advanced level of development. For potency testing, in vitro alternatives largely comprise immunochemical methods that assess several, but not all critical vaccine properties. One-to-one replacement by in vitro alternatives is not always possible and a combination of methods may be required.

A detailed analysis of innate and adaptive immune responsiveness upon infection with Salmonella enterica serotype Enteritidis in young broiler chickens.

Salmonella enterica serotype Enteritidis (SE) is a zoonotic pathogen which causes foodborne diseases in humans as well as severe disease symptoms in young chickens. More insight in innate and adaptive immune responses of chickens to SE infection is needed to understand elimination of SE. Seven-day-old broiler chickens were experimentally challenged with SE and numbers and responsiveness of innate and adaptive immune cells as well as antibody titers were assessed. SE was observed in the ileum and spleen of SE-infected chickens at 7 days post-infection (dpi). At 1 dpi numbers of intraepithelial cytotoxic CD8+ T cells were significantly increased alongside numerically increased intraepithelial IL-2Rα+ and 20E5+ natural killer (NK) cells at 1 and 3 dpi. At both time points, activation of intraepithelial and splenic NK cells was significantly enhanced. At 7 dpi in the spleen, presence of macrophages and expression of activation markers on dendritic cells were significantly increased. At 21 dpi, SE-induced proliferation of splenic CD4+ and CD8+ T cells was observed and SE-specific antibodies were detected in sera of all SE-infected chickens. In conclusion, SE results in enhanced numbers and activation of innate cells and we hypothesized that in concert with subsequent specific T cell and antibody responses, reduction of SE is achieved. A better understanding of innate and adaptive immune responses important in the elimination of SE will aid in developing immune-modulation strategies, which may increase resistance to SE in young broiler chickens.

Glucose Oligosaccharide and Long-Chain Glucomannan Feed Additives Induce Enhanced Activation of Intraepithelial NK Cells and Relative Abundance of Commensal Lactic Acid Bacteria in Broiler Chickens.

Restrictions on the use of antibiotics in the poultry industry stimulate the development of alternative nutritional solutions to maintain or improve poultry health. This requires more insight in the modulatory effects of feed additives on the immune system and microbiota composition. Compounds known to influence the innate immune system and microbiota composition were selected and screened in vitro, in ovo, and in vivo. Among all compounds, 57 enhanced NK cell activation, 56 increased phagocytosis, and 22 increased NO production of the macrophage cell line HD11 in vitro. Based on these results, availability and regulatory status, six compounds were selected for further analysis. None of these compounds showed negative effects on growth, hatchability, and feed conversion in in ovo and in vivo studies. Based on the most interesting numerical results and highest future potential feasibility, two compounds were analyzed further. Administration of glucose oligosaccharide and long-chain glucomannan in vivo both enhanced activation of intraepithelial NK cells and led to increased relative abundance of lactic acid bacteria (LAB) amongst ileum and ceca microbiota after seven days of supplementation. Positive correlations between NK cell subsets and activation, and relative abundance of LAB suggest the involvement of microbiota in the modulation of the function of intraepithelial NK cells. This study identifies glucose oligosaccharide and long-chain glucomannan supplementation as effective nutritional strategies to modulate the intestinal microbiota composition and strengthen the intraepithelial innate immune system.

Proteomic analysis of chicken bone marrow-derived dendritic cells in response to an inactivated IBV + NDV poultry vaccine.

Inactivated poultry vaccines are subject to routine potency testing for batch release, requiring large numbers of animals. The replacement of in vivo tests for cell-based alternatives can be facilitated by the identification of biomarkers for vaccine-induced immune responses. In this study, chicken bone marrow-derived dendritic cells were stimulated with an inactivated vaccine for infectious bronchitis virus and Newcastle disease virus, as well as inactivated infectious bronchitis virus only, and lipopolysaccharides as positive control, or left unstimulated for comparison with the stimulated samples. Next, the cells were lysed and subjected to proteomic analysis. Stimulation with the vaccine resulted in 66 differentially expressed proteins associated with mRNA translation, immune responses, lipid metabolism and the proteasome. For the eight most significantly upregulated proteins, mRNA expression levels were assessed. Markers that showed increased expression at both mRNA and protein levels included PLIN2 and PSMB1. Stimulation with infectious bronchitis virus only resulted in 25 differentially expressed proteins, which were mostly proteins containing Src homology 2 domains. Stimulation with lipopolysaccharides resulted in 118 differentially expressed proteins associated with dendritic cell maturation and antimicrobial activity. This study provides leads to a better understanding of the activation of dendritic cells by an inactivated poultry vaccine, and identified PLIN2 and PSMB1 as potential biomarkers for cell-based potency testing.

Elephant Endotheliotropic Herpesvirus Is Omnipresent in Elephants in European Zoos and an Asian Elephant Range Country.

Elephant endotheliotropic herpesviruses (EEHVs) may cause acute, often lethal, hemorrhagic disease (EEHV-HD) in young elephants. Prevalence of EEHV in different elephant populations is still largely unknown. In order to improve diagnostic tools for the detection of EEHV infections and to obtain insight into its spread among elephants, we developed novel ELISAs based on EEHV1A gB and gH/gL. Performance of the ELISAs was assessed using sera from 41 European zoo elephants and 69 semi-captive elephants from Laos, one of the Asian elephant range countries. Sera from all (sub)adult animals tested (≥5 years of age) showed high reactivity with both gB and gH/gL, indicating that EEHV prevalence has been highly underestimated so far. Reactivity towards the antigens was generally lower for sera of juvenile animals (1 > 5 years). Only one (juvenile) animal, which was sampled directly after succumbing to EEHV-HD, was found to be seronegative for EEHV. The two other EEHV-HD cases tested showed low antibody levels, suggesting that all three cases died upon a primary EEHV infection. In conclusion, our study suggests that essentially all (semi-)captive (sub)adult elephants in European zoos and in Laos carry EEHV, and that young elephants with low antibody levels are at risk of dying from EEHV-HD.

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

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

Analysis of chicken intestinal natural killer cells, a major IEL subset during embryonic and early life.

Restrictions on antimicrobials demand alternative strategies to improve broiler health, such as supplying feed additives which stimulate innate immune cells like natural killer (NK) cells. The main objective of this study was to characterize intestinal NK cells in broiler chickens during embryonic and early life and compare these to NK cells in spleen, blood and bone marrow. Also T-cell subsets were determined. The majority of intestinal NK cells expressed IL-2Rα rather than 20E5 and 5C7, and showed low level of activation. Within intestinal NK cells the activation marker CD107 was mostly expressed on IL-2Rα+ cells while in spleen and blood 20E5+ NK cells primarily expressed CD107. High percentages of intestinal CD8αα+, CD8αß+ and from 2 weeks onward also gamma delta T cells were found. Taken together, we observed several intestinal NK subsets in broiler chickens. Differences in NK subsets were mostly observed between organs, rather than differences over time. Targeting these intestinal NK subsets may be a strategy to improve immune-mediated resistance in broiler chickens.