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Elephant endotheliotropic herpesvirus (EEHV) causes lethal hemorrhagic disease (HD) in Asian and African elephants. Although rapid detection of viremia and supportive treatments may improve survival rates, an effective vaccine would mitigate the devastating effects of this virus. In elephants, chronic infection with EEHV leads to adaptive immunity against glycoproteins gB and gH/gL, the core entry machinery for most herpesviruses. We previously evaluated two EEHV gB vaccines in mice but not a gH/gL vaccine. Here, we found that inoculation of mice with an adjuvanted EEHV gH/gL subunit vaccine induced a significant antibody response that was similar to the response observed in elephants chronically infected with EEHV. Moreover, the gH/gL heterodimer elicited polyfunctional T cells with a Th1 phenotype but no detectable Th2 response. These results suggest that gH/gL, possibly in combination with gB, may be suitable immunogens for a vaccine comprising herpesvirus glycoproteins that are known to mediate cell entry and infection., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Paul D. Ling reports financial support was provided by International Elephant Foundation. Paul D. Ling reports financial support was provided by Houston Zoo. Tabitha E. Hoornweg reports financial support was provided by Named Fund Friends of VetMed. Victor P. M. G. Rutten reports financial support was provided by Named Fund Friends of VetMed. Cornelis A. M. de Haan reports financial support was provided by Named Fund Friends of VetMed. Jennifer L. Spencer Clinton reports financial support was provided by National Institutes of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The Esx-1 family proteins of the Type VII secretion systems of Mycobacterium bovis and Mycobacterium tuberculosis have been assessed and are frequently used as candidates for tuberculosis (TB) diagnosis in both humans and animals. The presence of ESAT-6 and CFP 10 proteins, which are the most immunogenic proteins of the Esx-1 system and have been widely investigated for the immunodiagnosis of tuberculosis, in some Mycobacteriaceae and in Mycobacterium leprae , poses limitations for their use in specific diagnoses of TB. As such, to improve the specificity of the ESAT-6/CFP 10-based cell-mediated immunity (CMI) assays, other proteins encoded by genes within and outside the RD 1 region of the esx-1 locus have been evaluated as candidate antigens for CMI, as well as to investigate humoral responses in combination with ESAT-6 and or CFP 10, with varying specificity and sensitivity results. Hence, in this study, we evaluated various non-tuberculous mycobacteria (NTM), Mycolicibacterium , Mycolicibacter and Mycobacteroides species genomes available on the NCBI database for the presence and composition of the RD1 region of the esx-1 locus. In addition, we also assayed by polymerase chain reaction (PCR) and sequencing of Mycobacteriaceae available in our culture collection for the presence and sequence diversity of esxA and esxB genes encoding ESAT-6 and CFP 10, respectively. Whole genome sequence (WGS) data analysis revealed the presence of RD 1 gene orthologs in 70 of the over 100 published genomes of pathogenic and non- pathogenic Mycobcteriaceae other than tuberculosis. Among species evaluated from our culture collection, in addition to earlier reports of the presence of esxA and esxB in certain Mycolicibacterium , Mycolicibacterium septicum/peregrinum , Mycolicibacterium porcinum and Mycobacterium sp. N845T were also found to harbour orthologs of both genes. Orthologs of esxA only were detected in Mycobacterium brasiliensis , Mycolicibacterium elephantis and Mycolicibacterium flouroantheinivorans , whereas in Mycolicibacter engbackii , Mycolicibacterium mageritense and Mycobacterium paraffinicum , only esxB orthologs were detected. A phylogenetic analysis based on esxA and esxB sequences separated slow-growing from rapidly growing bacteria. These findings strengthen previous suggestions that esxA and esxB may be encoded in the majority of Mycobacteriaceae . The role of the Esx-1 system in both pathogenic and non-pathogenic Mycobacteriaceae needs further investigation, as these species may pose limitations to immunological assays for TB.

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.

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., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 immune‑related 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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lyu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2022 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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-1β production as well as in the uptake of inactivated Leptospira in canine moDC. Furthermore, polymyxin B only partially inhibited IL-1β 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 ., Competing Interests: Authors EP and AS were employed by company Intravacc. Author AN was affiliated with company Intravacc at the time of study but was not formally employed. The remaining 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 Novak, Pupo, van’t Veld, Rutten, Broere and Sloots.)

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., 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 Geervliet, de Vries, Jansen, Rutten, van Hees, Wen, Skovgaard, Antonello, Savelkoul, Smidt, Tijhaar and Wells.)

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., (© 2022. The Author(s).)

This study aimed to investigate effects of transport age of calves (14 vs. 28 d), and of calf and dam characteristics, on immunoglobulin titers and hematological variables of veal calves. Calves (n = 683) were transported to a veal farm at 14 or 28 d of age. Natural antibodies N-IgG, N-IgM, and N-IgA against phosphorylcholine conjugated to bovine serum albumin (PC-BSA) were measured in serum of the dams 1 wk before calving and in first colostrum. These antibodies were also measured in serum of calves 1 wk after birth, 1 d before transport, and in wk 2 and 10 posttransport at the veal farm. Hematological variables were assessed in calves 1 d before transport and in wk 2 posttransport. One day before transport, titers of N-IgG, N-IgM, N-IgA, and neutrophil counts were higher, and lymphocyte counts were lower in 14-d-old calves compared with 28-d-old calves. In wk 2 at the veal farm, calves transported at 14 d of age had higher N-IgG titers and neutrophil counts, but lower N-IgM and N-IgA titers, and lymphocyte counts than calves transported at 28 d. In wk 1 and 1 d before transport, N-Ig in calves were positively related to N-Ig in colostrum. In wk 2 and 10 at the veal farm, N-IgG in calves was positively related to N-IgG in colostrum. The N-IgG titers in calves at the dairy farm were negatively related to the likelihood of being individually treated with antibiotics or other medicines at the veal farm. Our results suggest that calves transported to the veal farm at 28 d of age showed a more advanced development of their adaptive immunity than calves transported at 14 d of age. Quality of colostrum might have long-term consequences for N-IgG titers and immunity in veal calves., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

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.

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., 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 Angkwanish, Vernooij, Sirimalaisuwan, Charernpan, Nielen and Rutten.)

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.

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., (© 2021. The Author(s).)

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.

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.

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)., Competing Interests: The authors have declared that no competing interests exist.

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.

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 + , CD8αβ + 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., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Studies in mammals, including chickens, have shown that the development of the immune system is affected by interactions with intestinal microbiota. Early life microbial colonization may affect the development of innate and adaptive immunity and may contribute to lasting effects on health and resilience of broiler chickens. We inoculated broiler chickens with adult-derived-microbiota (AM) to investigate their effects on intestinal microbiota composition and natural killer (NK) cells, amongst other immune cells. We hypothesized that AM inoculation directly upon hatch (day 0) would induce an alteration in microbiota composition shortly after hatch, and subsequently affect (subsets of) intestinal NK cells and their activation. Microbiota composition of caecal and ileal content of chickens of 1, 3, 7, 14, 21, and 35 days of age was assessed by sequencing of 16S ribosomal RNA gene amplicons. In parallel, subsets and activation of intestinal NK cells were analyzed by flow cytometry. In caecal content of 1- and 3-day-old AM chickens, a higher alpha-diversity (Faith's phylogenetic diversity) was observed compared to control chickens, whereas ileal microbiota were unaffected. Regarding beta-diversity, caecal microbiota profiles could be clustered into three distinct community types. Cluster A represented caecal microbiota of 1-day-old AM chickens and 1- and 3-day-old control chickens. Cluster B included microbiota of seven of eight 3- and 7-day-old AM and 7-day-old control chickens, and cluster C comprised microbiota of all chickens of 14-days and older, independent of inoculation. In 3-day-old AM chickens an increase in the percentages of intestinal IL-2Rα + NK cells and activated NK cells was observed compared to control chickens of the same age. In addition, an increase in relative numbers of intestinal cytotoxic CD8αα + T cells was observed in 14- and 21-day-old AM chickens. Taken together, these results indicate that early exposure to AM shapes and accelerates the maturation of caecal microbiota, which is paralleled by an increase in IL-2Rα + NK cells and enhanced NK cell activation. The observed association between early life development of intestinal microbiota and immune system indicates possibilities to apply microbiota-targeted strategies that can accelerate maturation of intestinal microbiota and strengthen the immune system, thereby improving the health and resilience of broiler chickens., (Copyright © 2020 Meijerink, Kers, Velkers, van Haarlem, Lamot, de Oliveira, Smidt, Stegeman, Rutten and Jansen.)

High-quality vaccines are crucial to prevent infectious disease outbreaks in the poultry industry. In vivo vaccination tests are routinely used to test poultry vaccines for their potency, i.e., their capacity to induce protection against the targeted diseases. A better understanding of how poultry vaccines activate immune cells will facilitate the replacement of in vivo potency tests for in vitro assays. Using the chicken macrophage-like HD11 cell line as a model to evaluate innate immune responses, the current explorative study addresses the immunostimulatory capacity of an inactivated multivalent vaccine for infectious bronchitis, Newcastle disease, egg-drop syndrome, and infectious coryza. The vaccine stimulated HD11 cells to produce nitric oxide and to express pro-inflammatory cytokines IL-1β , TNF, and IL-12p40, chemokines CXCLi1 and CXCLi2 , and the anti-inflammatory cytokine IL-10 , but only when inactivated Avibacterium paragallinarum , the causative agent of infectious coryza, was present. Lipopolysaccharides from Avibacterium paragallinarum were crucial for the production of nitric oxide and expression of IL-1β and CXCLi1 . The described immune parameters demonstrate the capacity of this multivalent vaccine to activate innate immune cells and may in the future, combined with antigen quantification methods, contribute to vaccine quality testing in vitro, hence the replacement of current in vivo vaccination tests.

Intramammary infections (IMI) with Staphylococcus aureus are a common cause of bovine mastitis and can result in both clinical (CM) or subclinical mastitis (SCM). Although bacterial isolates of S. aureus differ in their virulence potential it is largely unclear which bacterial virulence factors are responsible for increased clinical severity. We performed a genome wide association study and used a generalized linear mixed model to investigate the correlation between gene carriage, lineage and clinical outcome of IMI in a collection of S. aureus isolates from cattle with CM (n = 125) and SCM (n = 151) from 11 European countries. An additional aim was to describe the genetic variation of bovine S. aureus in Europa. The dominant lineages in our collection were clonal complex (CC) 151 (81/276, 29.3%), CC97 (54/276, 19.6%), CC479 (32/276, 11.6%) and CC398 (19/276, 6.9%). Virulence and antimicrobial resistance (AMR) gene carriage was highly associated with CC. Among a selection of nine virulence and AMR genes, CC151, CC479 and CC133 carried more virulence genes than other CCs, and CC398 was associated with AMR gene carriage. Whereas CC151, CC97 were widespread in Europe, CC479, CC398 and CC8 were only found in specific countries. Compared to CC151, CC479 was associated with CM rather than SCM (OR 3.62; 95% CI 1.38-9.50) and the other CCs were not. Multiple genes were associated with CM, but due to the clustering within CC of carriage of these genes, it was not possible to differentiate between the effect of gene carriage and CC on clinical outcome of IMI. Nevertheless, this study demonstrates that characterization of S. aureus CC and virulence genes helps to predict the likelihood of the occurrence of CM following S. aureus IMI and highlights the potential benefit of diagnostics tools to identify S. aureus CC during bovine mastitis.

Piglets are susceptible to infections in early life and around weaning due to rapid environmental and dietary changes. A compelling target to improve pig health in early life is diet, as it constitutes a pivotal determinant of gut microbial colonization and maturation of the host's immune system. In the present study, we investigated how supplementation of yeast-derived β-glucans affects the gut microbiota and immune function pre- and post-weaning, and how these complex systems develop over time. From day two after birth until two weeks after weaning, piglets received yeast-derived β-glucans or a control treatment orally and were subsequently vaccinated against Salmonella Typhimurium. Faeces, digesta, blood, and tissue samples were collected to study gut microbiota composition and immune function. Overall, yeast-derived β-glucans did not affect the vaccination response, and only modest effects on faecal microbiota composition and immune parameters were observed, primarily before weaning. This study demonstrates that the pre-weaning period offers a 'window of opportunity' to alter the gut microbiota and immune system through diet. However, the observed changes were modest, and any long-lasting effects of yeast-derived β-glucans remain to be elucidated.

The pathogenesis of many inflammatory diseases is associated with the uncontrolled activation of nuclear factor kappa B (NF-κB) in macrophages. Previous studies have shown that in various cell types, heat shock protein 70 (Hsp70) plays a crucial role in controlling NF-κB activity. So far, little is known about the role of Hsp70 in canine inflammatory processes. In this study we investigated the potential anti-inflammatory effects of Hsp70 in canine macrophages as well as the mechanisms underlying these effects. To this end, a canine macrophage cell line was stressed with arsenite, a chemical stressor, which upregulated Hsp70 expression as detected by flow cytometry and qPCR. A gene-edited version of this macrophage cell line lacking inducible Hsp70 was generated using CRISPR-Cas9 technology. To determine the effects of Hsp70 on macrophage inflammatory properties, arsenite-stressed wild-type and Hsp70 knockout macrophages were exposed to lipopolysaccharide (LPS), and the expression of the inflammatory cytokines IL-6, IL-1β and tumor necrosis factor-α (TNF-α) and levels of phosphorylated NF-κB were determined by qPCR and Western Blotting, respectively. Our results show that non-toxic concentrations of arsenite induced Hsp70 expression in canine macrophages; Hsp70 upregulation significantly inhibited the LPS-induced expression of the pro-inflammatory mediators TNF-α and IL-6, as well as NF-κB activation in canine macrophages. Furthermore, the gene editing of inducible Hsp70 by CRISPR-Cas9-mediated gene editing neutralized this inhibitory effect of cell stress on NF-κB activation and pro-inflammatory cytokine expression. Collectively, our study reveals that Hsp70 may regulate inflammatory responses through NF-κB activation and cytokine expression in canine macrophages.

In early life and around weaning, pigs are at risk of developing infectious diseases which compromise animal welfare and have major economic consequences for the pig industry. A promising strategy to enhance resistance against infectious diseases is immunomodulation by feed additives. To assess the immune stimulating potential of feed additives in vitro, bone marrow-derived dendritic cells were used. These cells play a central role in the innate and adaptive immune system and are the first cells encountered by antigens that pass the epithelial barrier. Two different feed additives were tested on dendritic cells cultured from fresh and cryopreserved bone marrow cells; a widely used commercial feed additive based on yeast-derived β-glucans and the gram-negative probiotic strain E. coli Nissle 1917. E. coli Nissle 1917, but not β-glucans, induced a dose-dependent upregulation of the cell maturation marker CD80/86, whereas both feed additives induced a dose-dependent production of pro- and anti-inflammatory cytokines, including TNFα, IL-1β, IL-6 and IL-10. Furthermore, E. coli Nissle 1917 consistently induced higher levels of cytokine production than β-glucans. These immunomodulatory responses could be assessed by fresh as well as cryopreserved in vitro cultured porcine bone marrow-derived dendritic cells. Taken together, these results demonstrate that both β-glucans and E. coli Nissle 1917 are able to enhance dendritic cell maturation, but in a differential manner. A more mature dendritic cell phenotype could contribute to a more efficient response to infections. Moreover, both fresh and cryopreserved bone marrow-derived dendritic cells can be used as in vitro pre-screening tools which enable an evidence based prediction of the potential immune stimulating effects of different feed additives., Competing Interests: The authors have declared that no competing interests exist.

The endemic presence of bovine tuberculosis (BTB) in African buffaloes in South Africa has severe consequences for BTB control in domestic cattle, buffalo ranching and wildlife conservation, and poses a potential risk to public health. This study determined the BTB prevalence in free-ranging buffaloes in two game reserves and assessed the influence of the prevalence of mycobacterial infections on the performance of a commercial cattle-specific serological assay for BTB (TB ELISA). Buffaloes (n = 997) were tested with the tuberculin skin test and TB ELISA; a subset (n = 119) was tested longitudinally. Culture, PCR and sequencing were used to confirm infection with M. bovis and/or non-tuberculous mycobacteria (NTM). Prevalence of BTB, but not NTM, influenced the TB ELISA performance. Multiple testing did not increase test confidence. The findings strongly illustrate the need for development of novel assays that can supplement existing assays for a more comprehensive testing scheme for BTB in African buffaloes., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Dysregulation of retinal pigment epithelium (RPE) cells is the main cause of a variety of ocular diseases. Potentially heat shock proteins, by preventing molecular and cellular damage and modulating inflammatory disease, may exert a protective role in eye disease. In particular, the inducible form of heat shock protein 70 (Hsp70) is widely upregulated in inflamed tissues, and in vivo upregulation of Hsp70 expression by HSP co-inducing compounds has been shown to be a potential therapeutic strategy for inflammatory diseases. In order to gain further understanding of the potential protective effects of Hsp70 in RPE cells, we developed a method for isolation and culture of canine RPE cells. Identity of RPE cells was confirmed by detection of its specific marker, RPE65, in qPCR, flow cytometry, and immunocytochemistry analysis. The ability of RPE cells to express Hsp70 upon experimental induction of cell stress, by arsenite, was analyzed by flow cytometry. Finally, in search of a potential Hsp70 co-inducer, we investigated whether the compound leucinostatin could enhance Hsp70 expression in stressed RPE cells. Canine RPE cells were isolated and cultured successfully. Purity of cells that strongly expressed RPE65 was over 90%. Arsenite-induced stress led to a time- and dose-dependent increase in Hsp70 expression in canine RPE cells in vitro. In addition, leucinostatin, which enhanced heat shock factor-1-induced transcription from the heat shock promoter in DNAJB1-luc-O23 reporter cell line, also enhanced Hsp70 expression in arsenite-stressed RPE cells, in a dose-dependent fashion. These findings demonstrate that leucinostatin can boost Hsp70 expression in canine RPE cells, most likely by activating heat shock factor-1, suggesting that leucinostatin might be applied as a new co-inducer for Hsp70 expression.

Research in chickens has been fundamental for the discovery of basic aspects of the immune system and has led to an interest in the in-depth characterization of avian immune cell types including dendritic cells (DCs). The in vitro generation and expansion of chicken bone marrow-derived DCs (chBMDCs) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) has provided a way to study chicken DCs, which are only present at limited cell numbers in vivo . This method has been employed to study the interactions between chicken DCs and pathogens or vaccines. However, a detailed characterization of the chBMDC culture is still lacking. In the present study, we performed an elaborate phenotypical and functional analysis of the chBMDC culture and addressed its heterogeneity. After 8 days of culture, chBMDCs comprised major histocompatibility complex class II (MHC-II) low and MHC-II high subsets with different morphologies. Compared with MHC-II low chBMDCs, the MHC-II high subset showed a more mature phenotype, with higher expressions of CD1.1, CD40, CD80, CCR7, and CD83, and a relatively low opsonophagocytic capacity. Nevertheless, MHC-II high chBMDCs did not show an increased capacity to induce T-cell proliferation. Therefore, MHC-II high chBMDCs were found to be semi-mature. Interestingly, the presence of the semi-mature MHC-II high chBMDC subset reduced when cells were cultured in the presence of IL-4. Finally, prolonged cell culture after fluorescence-activated cell sorting (FACS) converted the semi-mature MHC-II high subset back into the immature phenotype of the MHC-II low subset, demonstrating plasticity of their maturation state. This detailed characterization explained the heterogeneity of the chBMDC culture by the simultaneous presence of immature and semi-mature chBMDC subsets, in addition to cells without features of antigen-presenting cells. Our findings are instrumental for the interpretation of experiments using the chBMDC culture in past and future research by providing insights into its phenotypically and functionally distinct cell types., (Copyright © 2020 van den Biggelaar, Arkesteijn, Rutten, van Eden and Jansen.)

Bovine mastitis is a costly disease to the dairy industry and intramammary infections (IMI) with Staphylococcus aureus are a major cause of mastitis. Staphylococcus aureus strains responsible for mastitis in cattle predominantly belong to ruminant-associated clonal complexes (CCs). Recognition of pathogens by bovine mammary epithelial cells (bMEC) plays a key role in activation of immune responsiveness during IMI. However, it is still largely unknown to what extent the bMEC response differs according to S. aureus CC. The aim of this study was to determine whether ruminant-associated S. aureus CCs differentially activate bMEC. For this purpose, the immortalized bMEC line PS was stimulated with S. aureus mastitis isolates belonging to four different clonal complexes (CCs; CC133, CC479, CC151 and CC425) and interleukin 8 (IL-8) release was measured as indicator of activation. To validate our bMEC model, we first stimulated PS cells with genetically modified S. aureus strains lacking (protein A, wall teichoic acid (WTA) synthesis) or expressing (capsular polysaccharide (CP) type 5 or type 8) factors expected to affect S. aureus recognition by bMEC. The absence of functional WTA synthesis increased IL-8 release by bMEC in response to bacterial stimulation compared to wildtype. In addition, bMEC released more IL-8 after stimulation with S. aureus expressing CP type 5 compared to CP type 8 or a strain lacking CP expression. Among the S. aureus lineages, isolates belonging to CC133 induced a significantly stronger IL-8 release from bMEC than isolates from the other CCs, and the IL-8 response to CC479 was higher compared to CC151 and CC425. Transcription levels of IL-8, tumor necrosis factor alpha (TNFα), serum amyloid A3 (SAA3), Toll-like receptor (TLR)-2 and nuclear factor κB (NF-κB) in bMEC after bacterial stimulation tended to follow a similar pattern as IL-8 release, but there were no significant differences between the CCs. This study demonstrates a differential activation of bMEC by ruminant-associated CCs of S. aureus , which may have implications for the severity of mastitis during IMI by S. aureus belonging to these lineages.

Staphylococcus aureus is an important mastitis pathogen, causing both clinical mastitis (CM) and subclinical mastitis (SCM) in small ruminants. In general, CM has a low incidence in sheep and goats but can be very severe and costly. In contrast, subclinical mastitis (SCM) is common but is associated with less cost. For both sheep and goats, S. aureus is the main cause of CM and is associated with SCM cases with a high SCC. Recently, specific lineages of S. aureus have been identified that are associated with CM rather than SCM in dairy cows. It is unknown whether specific S. aureus lineages are associated with CM in goats and sheep. The aim of this study was to compare the clonal complex (CC), staphylococcal protein A (spa) type, leukocidin lukM-lukF' presence, and potential to produce LukMF' in vitro between CM and SCM S. aureus mastitis isolates obtained from sheep and goats. Differences between isolates from different host species were also compared. Ovine (CM, n = 12; SCM, n = 29) and caprine (CM, n = 14; SCM, n = 30) isolates were obtained from 8 sheep flocks and 8 goat herds in the Netherlands. Overall, the isolates belonged to CC133 (85%), CC398 (7%), CC425 (5%), and CC45 (2%). Seventeen spa types were found, including 6 novel types; the predominant types were t2678 (34%), t544 (18%), and t3583 (18%). Although CC133 was dominant among both sheep and goat isolates, spa type CC133/t2678 was associated with ovine isolates, whereas CC133/t544 and CC133/t3583 were found mostly in goats. The presence of lukM-lukF' among the S. aureus isolates was high (87%), especially in CC133 (96%) and CC425 (100%), but the genes were absent in CC45 and CC398. In vitro-cultured lukM-lukF'-positive isolates produced LukM (71 out of 74 positive isolates tested) in the range of 0.4 to 5.0 µg/mL. Interestingly, the goat-associated lineages CC133/t544 and CC133/t3583 produced more LukM in vitro than the sheep-associated CC133/t2678. We found no difference in LukMF' production potential between CM and SCM isolates. In sheep as well as in goats, no association was found between genotype and CM or SCM, demonstrating that the same lineages of S. aureus are responsible for both CM and SCM. These results suggest that subclinically infected animals in a herd or flock likely act as the reservoir of S. aureus causing CM. This highlights the importance of early identification and control of SCM and suggests that controlling SCM within a herd is an effective intervention to prevent CM in small ruminants., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: Elephant endotheliotropic herpesviruses (EEHV) can cause an acute highly fatal hemorrhagic disease in young Asian elephants (Elephas maximus), both ex situ and in situ. Amongst eight EEHV types described so far, type 1 (subtype 1A and 1B) is the predominant disease-associated type. Little is known about routes of infection and pathogenesis of EEHV, and knowledge of disease prevalence, especially in range countries, is limited., Methods: A large cross-sectional serological survey was conducted in captive elephants (n = 994) throughout Thailand using an EEHV-1A glycoprotein B protein antigen specific antibody ELISA., Results: Antibody seroprevalence was 42.3%, with 420 of 994 elephants testing positive. Associations between seropositivity and potential risk factors for EEHV infection were assessed and included: elephant age, sex, camp cluster size, management type (extensive versus intensive), sampling period (wet vs. dry season) and location of camp (region). Univariable regression analysis identified management system and region as risk factors for the presence of EEHV antibodies in elephants, with region being significant in the final multivariable regression model. Prevalence was highest in the North region of the country (49.4%)., Conclusions: This study produced baseline serological data for captive elephants throughout Thailand, and showed a significant EEHV burden likely to be maintained in the captive population.

Canine atopic dermatitis is a genetically predisposed inflammatory and pruritic allergic skin disease that is often complicated by (secondary) bacterial and fungal (yeast) infections. High-throughput DNA sequencing was used to characterize the composition of the microbiome (bacteria and fungi) inhabiting specific sites of skin in healthy dogs and dogs with atopic dermatitis (AD) before and after topical antimicrobial treatment. Skin microbiome samples were collected from six healthy control dogs and three dogs spontaneously affected by AD by swabbing at (non-) predilection sites before, during and after treatment. Bacteria and fungi were profiled by Illumina sequencing of the 16S ribosomal RNA gene of bacteria (16S) and the internally transcribed spacer of the ribosomal gene cassette in fungi (ITS). The total cohort of dogs showed a high diversity of microbes on skin with a strong individual variability of both 16S and ITS profiles. The genera of Staphylococcus and Porphyromonas were dominantly present both on atopic and healthy skin and across all skin sites studied. In addition, bacterial and fungal alpha diversity were similar at the different skin sites. The topical antimicrobial treatment increased the diversity of bacterial and fungal compositions in course of time on both AD and healthy skin., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Keratinocytes (KC) are the main cellular components of the stratum corneum that constitutes a solid physical skin barrier representing the first line of defense against pathogens. Moreover, KC are potent producers of inflammatory mediators and antimicrobial peptides (AMP) when activated through their pattern recognition receptors. In atopic dermatitis (AD) the protective skin barrier may be compromised due to barrier disruption, secondary infection and accelerated secretion of inflammatory cytokines which may also affect AMP expression in the skin. In the present study, we addressed the responses of a canine KC cell line upon exposure to Staphylococcus pseudintermedius, typically found on canine atopic skin during secondary infections, and stimulation by individual AD-associated ligands and cytokines. All stimuli induced a significant increase in expression of the pro-inflammatory cytokine genes tumor necrosis factor (TNF)-α and interleukin (IL)-8, but with different kinetics. Limited effects were observed on AMP gene expression except for K9CATH which was significantly upregulated upon bacterial infection but with none of the individual AD-associated ligands. Interestingly, K9CATH possessed antimicrobial activity towards Staphylococcus pseudintermedius, indicating that K9CATH expression is a specific defense reaction towards bacterial infection and not part of a general pro-inflammatory profile of KC., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Staphylococcus aureus is a contagious, opportunistic pathogen that causes clinical or subclinical mastitis in dairy cattle. The genetic background and antimicrobial resistance of isolates from Ethiopian dairy farms has not been studied. Therefore, the aim of this study was to characterize S. aureus from Ethiopian hand milked dairy cows, by spa, MLST and virulence factor typing, and by assessment of antimicrobial susceptibility. A total of 79 S. aureus isolates from intramammary infections was studied. A PCR was used to detect lukM-lukF' and pvl genes encoding the bovine and human associated bi-component leukocidins, and the toxic shock syndrome toxin gene-1 (tst). Antimicrobial susceptibility was determined using the broth microdilution method., Results: Twenty different spa types were identified, most isolates were t042 (58%), and the closely related t15786 (11%). The proportion of isolates positive for lukM-lukF', tst and pvl was low at 0.04, 0.10 and 0.09 respectively, with lukM-lukF' often co-occurring with tst, but not with pvl. Methicillin-resistance was not found, but resistance to penicillin/ampicillin (86%) and tetracycline (54%) was very common., Conclusions: We found a high degree of relatedness among bovine S. aureus isolates in North-Western Ethiopia, suggesting contagious within and between farm transmission of strains that are often resistant to commonly used antimicrobials. This highlights the need for effective preventive measures that aim at limiting transmission of bacteria rather than using antimicrobials to control S. aureus mastitis in Ethiopia.

Some species of non-tuberculous mycobacteria (NTM) have been reported to be opportunistic pathogens of animals and humans. Recently there has been an upsurge in the number of cases of NTM infections, such that some NTM species are now recognized as pathogens of humans and animals. From a veterinary point of view, the major significance of NTM is the cross-reactive immune response they elicit against Mycobacterium bovis antigens, leading to misdiagnosis of bovine tuberculosis. Four NTM isolates were detected from a bovine nasal swab, soil and water, during an NTM survey in South Africa. These were all found using 16S rRNA gene sequence analysis to be closely related to Mycobacterium moriokaense. The isolates were further characterised by sequence analysis of the partial fragments of hsp65, rpoB and sodA. The genome of the type strain was also elucidated. Gene (16S rRNA, hsp65, rpoB and sodA) and protein sequence data analysis of 6 kDa early secretory antigenic target (ESAT 6) and 10 kDa culture filtrate protein (CFP-10) revealed that these isolates belong to a unique Mycobacterium species. Differences in phenotypic and biochemical traits between the isolates and closely related species further supported that these isolates belong to novel Mycobacterium species. We proposed the name Mycobacterium komaniense sp. nov. for this new species. The type strain is GPK 1020 T (=CIP 110823T=ATCC BAA-2758).

In the last decades, many regional and country-wide control programmes for Johne's disease (JD) were developed due to associated economic losses, or because of a possible association with Crohn's disease. These control programmes were often not successful, partly because management protocols were not followed, including the introduction of infected replacement cattle, because tests to identify infected animals were unreliable, and uptake by farmers was not high enough because of a perceived low return on investment. In the absence of a cure or effective commercial vaccines, control of JD is currently primarily based on herd management strategies to avoid infection of cattle and restrict within-farm and farm-to-farm transmission. Although JD control programmes have been implemented in most developed countries, lessons learned from JD prevention and control programmes are underreported. Also, JD control programmes are typically evaluated in a limited number of herds and the duration of the study is less than 5 year, making it difficult to adequately assess the efficacy of control programmes. In this manuscript, we identify the most important gaps in knowledge hampering JD prevention and control programmes, including vaccination and diagnostics. Secondly, we discuss directions that research should take to address those knowledge gaps., (© 2017 Blackwell Verlag GmbH.)

Mycobacterium bovis (M.bovis) is the main causative agent for bovine tuberculosis (BTB) and can also be the cause of zoonotic tuberculosis in humans. In view of its zoonotic nature, slaughterhouse surveillance, potentially resulting in total or partial condemnation of the carcasses and organs, is conducted routinely. Spoligotyping, VNTR profiling, and whole genome sequencing (WGS) of M. bovis isolated from tissues with tuberculosis-like lesions collected from 14 cattle at Eritrea's largest slaughterhouse in the capital Asmara, were conducted.The 14 M. bovis isolates were classified into three different spoligotype patterns (SB0120, SB0134 and SB0948) and six VNTR profiles. WGS results matched those of the conventional genotyping methods and further discriminated the six VNTR profiles into 14 strains. Furthermore, phylogenetic analysis of the M. bovis isolates suggests two independent introductions of BTB into Eritrea possibly evolving from a common ancestral strain in Europe.This molecular study revealed the most important strains of M. bovis in Eritrea and their (dis)similarities with the strains generally present in East Africa and Europe, as well as potential routes of introduction of M. bovis. Though the sample size is small, the current study provides important information as well as platform for future in-depth molecular studies on isolates from both the dairy and the traditional livestock sectors in Eritrea and the region. This study provides information onthe origin of some of the M. bovis strains in Eritrea, its genetic diversity, evolution and patterns of spread between dairy herds. Such information is essential in the development and implementation of future BTB control strategy for Eritrea.

Accurate diagnosis of tuberculosis in cattle may be compromised in areas where there are high rates of exposure to environmental/non-tuberculous mycobacteria (NTM). This cross reaction of immune responses to Mycobacterium bovis antigens shared with NTMs can result in reduced specificity of commonly used diagnostic tests including tuberculin skin tests and the interferon gamma assay (IFN-ɣ). In this study we assessed the cross-reactive immune responses of M. bovis (infected) and NTM exposed animals to M. bovis and M. avium tuberculin, the ESAT6/CFP10 cocktail antigen, tuberculin derived from cultures of selected NTMs, and a panel of recombinant mycobacterium tuberculosis complex (MTBC) antigens sharing homology with orthologues in NTM. Gamma interferon (IFN-ɣ) responses were measured in whole blood cultures using the IFN-ɣ assay and the IFN-ɣ elispot assay on purified peripheral blood mononuclear cells (PBMC). We observed the expected strong IFN-ɣ response to PPD-B in the M. bovis infected animals that distinguished this group from non-infected NTM exposed cattle. The IFN-ɣ responses to PPD-N (M. nonchromogenicum), were relatively high in both infected and non-infected NTM exposed cattle, but were not significantly different to classify the true infection status of each group. The results indicated that the cross-reactive responses to PPD-B and/or PPD-A with PPD-N, likely arose from prior exposure to environmental non-tuberculous mycobacteria. The IFN-ɣ immune responses to the 10 R-Mag measured by the IFN-ɣ elispot assay revealed that three of the selected antigens, Rv3615 (ESpC), Rv0287 (esxG) and the ESAT6/CFP10, were immunogenic in the infected cattle, and distinguished the infected cattle from the non-infected NTM exposed animals. The combined data of PPDs and R-Mags derived from NTM mycobacteria may prove useful in future development of novel bTB diagnostic tests., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The aim of the current study was to assess the prevalence of bovine tuberculosis (BTB) in cattle, goats, and camels, and its zoonotic potential within the traditional livestock raising communities in four regions of Eritrea. The Single Intradermal Comparative Tuberculin Test (SICTT) as indicator of M. bovis infection was conducted on 1077 cattle, 876 goats, and 195 camels. To elucidate possible risk factors for BTB transmission between animals and its potential zoonotic implication, questionnaire based face-to-face interviews were conducted in households of which 232 raised cattle, 128 goats, and 29 camels., Results: The results of the SCITT were interpreted using the OIE standard (> 4 mm cut-off) for positive responses. In cattle, individual animal (n = 1077) and herd (n = 413) prevalences were 1.2% (n = 13) [Confidence Interval (CI) 95% CI, 1.0-1.3%] and 3.2% (n = 13) (95% CI, 3.0-3.4%), respectively. In goats (n = 876), none of the animals was positive. In camels, individual animal (n = 195) and herd (n = 70), BTB prevalences were 1.5% (n = 3) (95% CI,1.4-1.6%) and 2.9(n = 2) (95% CI, 0.9-4.6%), respectively. Overall, male animals were more at risk (OR = 2.6; 95% CI:1.0-8.7) when compared to females. Sharing of water points, introduction of new animals into herds and migration of animals over large distances were common events that may contribute to intra and inter-species transmission of BTB. Consumption of raw milk, lack of BTB transmission awareness, and low levels of education were common in the farming communities., Conclusion: The current study highlighted a low prevalence of M. bovis in cattle, goats and camels in extensive traditional livestock in Eritrea. Despite this, the spatial distribution of affected animals across most of the sampled regions and consumption of unpasteurized milk warrants surveillance, cautious and timely control measures for the disease.

The scientific community has reported several cases of microbes that exhibit elevated rates of antibiotic resistance in different regions of the planet. Due to this emergence of antimicrobial resistant microorganisms, the use of antibiotics as promoters of livestock animals' growth is being banned in most countries around the world. One of the challenges of agricultural immunology therefore is to find alternatives by modulating the immune system of animals in drug-independent safe food production systems. In this regard, in an effort to supplant antibiotics from bovine feeds, several alternatives were proposed including the use of immunomodulatory probiotics (immunobiotics). The purpose of this review is to provide an update of the status of the modulation of intestinal antiviral innate immunity of the bovine host by immunobiotics, and the beneficial impact of immunobiotics on viral infections, focused on intestinal epithelial cells (IECs). The results of our group, which demonstrate the capacity of immunobiotic strains to beneficially modulate Toll-like receptor 3-triggered immune responses in bovine IECs and improve the resistance to viral infections, are highlighted. This review provides comprehensive information on the innate immune response of bovine IECs against virus, which can be further investigated for the development of strategies aimed to improve defenses in the bovine host.

Intramammary infections in cattle resulting in mastitis have detrimental effects on cows' well-being, lifespan and milk production. In the host defense against S. aureus mastitis antibodies are thought to play an important role. To explore potential ways to increase antibody titers in the bovine mammary gland the effects of various adjuvants on the magnitude, isotype, and neutralizing capacity of antibodies produced following subcutaneous vaccine administration at different immunization sites were analyzed. In this study, α-toxoid was used as a model antigen and formulated in three different alum-based adjuvants: Alum-Saponin, Alum-Oil, and Alum-Saponin-Oil. Vaccines were administered near the suspensory ligament of the udder or in the lateral triangular area of the neck. At both immunization sites, immunization with α-toxoid in Alum-Saponin-Oil resulted in higher specific antibody titers in milk and serum as compared with Alum-Oil and Alum-Saponin, without favoring an IgG1, IgG2, or IgA response. Furthermore, the neutralizing capacity of milk serum and serum following immunization near the udder and in the neck was higher when Alum-Saponin-Oil was used as adjuvant compared with Alum-Oil and Alum-Saponin. Prime immunizations near the udder effectively increased both antibody isotype titers and neutralization titers, while prime plus boost immunizations were required to induce similar effects following immunization in the neck. Results indicate that subcutaneous administration of an Alum-Saponin-Oil based vaccine near the udder could be further explored for the development of a one-shot vaccination strategy to efficiently increase intramammary antibody responses.

Skin barrier disruption plays a role in the pathogenesis of atopic dermatitis (AD) in humans. However, little is known about skin barrier (dys-) function in Canine Atopic Dermatitis. The properties of lipids located in the outermost layer of the skin, the stratum corneum (SC) are considered to be important for the barrier. In the present study the lipid composition and lipid organization of the SC of AD dogs and control dogs were examined. The lipid composition of lesional AD skin as compared to control skin, showed a reduced free fatty acid level and a decreased ratio of ceramide[NS] C44/C34, in which C44 and C34 are the total numbers of carbon atoms of the sphingosine (S) and non-hydroxy (N) acyl chains. As a consequence of the observed changes in lipid composition in AD lesional skin the lamellar organization of lipids altered and a shift from orthorhombic to hexagonal lipid packing was monitored. Simultaneously an increased conformational disordering occurred. These changes are expected to compromise the integrity of the skin barrier. The C44/C34 chain length ratio of ceramide[NS] also showed a decreasing nonlinear relationship with the AD severity score (CADESI). Taken together, canine atopic skin showed alterations in SC lipid properties, similar to the changes observed in atopic dermatitis in humans, that correlated with a disruption of the skin barrier. Hence lipids play an important role in the pathogenesis of Canine Atopic Dermatitis., (Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.)

The aim of our study was to determine the association of selected potential risk factors with the presence of bovine tuberculosis (BTB) in dairy herds in Eritrea. A case-control study was conducted in the three major milk-producing regions of the country by stratified random sampling of 61 case and 65 control herds combined with completion of a standardized pretested questionnaire pertaining 36 relevant risk factors (variables). The variables were divided into two clusters, based on potential association with either "introduction" or "establishment" of BTB on the farms to elucidate association with incident or prevalent cases separately. Subsequent to univariable analysis of the 36 risk factors at herd level, 14 of these were offered to multivariable logistic regression models. Farms with higher numbers of cows, and those with concrete floors, were 3.6, and 7.5 times more at risk for presence of BTB, respectively, compared with their references. These findings will be useful as entry points for future informed decision-making towards BTB control and eradication programme in the country., (© 2017 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH.)

Mycobacterium bovis is the cause of tuberculosis (TB) in a wide range of species, including white rhinoceroses ( Ceratotherium simum ). Control of the disease relies on the indirect detection of infection by measuring pathogen-specific responses of the host. These are poorly described in the white rhinoceros and this study aimed to characterize the kinetics of immune responses to M. bovis infection in this species. Three white rhinoceroses were infected with M. bovis and their immune sensitization to this pathogen was measured monthly for 20 months. Cell-mediated immunity was characterized in whole blood samples as the differential release of interferon-gamma in response to bovine purified protein derivative (PPDb) and avian PPD (PPDa) as well as the release of this cytokine in response to the M. bovis proteins 6 kDa early secretory antigenic target (ESAT-6)/10 kDa culture filtrate protein (CFP-10). Humoral immunity was quantified as the occurrence or the magnitude of antibody responses to the proteins ESAT-6/CFP-10, MPB83, MPB83/MPB70, and PPDb. The magnitude and duration of immune reactivity varied between individuals; however, peak responses to these antigens were detected in all animals circa 5-9 months postinfection. Hereafter, they gradually declined to low or undetectable levels. This pattern was associated with limited TB-like pathology at postmortem examination and appeared to reflect the control of M. bovis infection following the development of the adaptive immune response. Measurement of these markers could prove useful for assessing the disease status or treatment of naturally infected animals. Moreover, immune responses identified in this study might be used to detect infection; however, further studies are required to confirm their diagnostic utility.

Conventional control and eradication strategies for bovine tuberculosis (BTB) face tremendous difficulties in developing countries; countries with wildlife reservoirs, a complex wildlife-livestock-human interface or a lack of veterinary and veterinary public health surveillance. Vaccination of cattle and other species might in some cases provide the only suitable control strategy for BTB, while in others it may supplement existing test-and-slaughter schemes. However, the use of live BCG has several limitations and the global rise of HIV/AIDS infections has furthermore warranted the exploration of inactivated vaccine preparations. The aim of this study was to compare the immune response profiles in response to parenteral vaccination with live BCG and two inactivated vaccine candidates in cattle. Twenty-four mixed breed calves (Bos taurus) aged 4-6 months, were allocated to one of four groups and vaccinated sub-cutaneously with live M. bovis BCG (Danish 1331), formalin-inactivated M. bovis BCG, heat-killed M. bovis or PBS/Montanide™ (control). Interferon-γ responsiveness and antibody production were measured prior to vaccination and at weekly intervals thereafter for twelve weeks. At nine weeks post-priming, animals were skin tested using tuberculins and MTBC specific protein cocktails and subsequently challenged through intranodular injection of live M. bovis BCG. The animals in the heat-killed M. bovis group demonstrated strong and sustained cell-mediated and humoral immune responses, significantly higher than the control group in response to vaccination, which may indicate a protective immune profile. Animals in this group showed reactivity to the skin test reagents, confirming good vaccine take. Lastly, although not statistically significant, recovery of BCG after challenge was lowest in the heat-killed M. bovis group. In conclusion, the parenteral heat-killed M. bovis vaccine proved to be clearly immunogenic in cattle in the present study, urging further evaluation of the vaccine in challenge studies using virulent M. bovis and assessment of vaccine efficacy in field conditions.

Failing immunological tolerance for critical self-antigens is the problem underlying most chronic inflammatory diseases of humans. Despite the success of novel immunosuppressive biological drugs, the so-called biologics, in the treatment of diseases such rheumatoid arthritis (RA) and type 1 diabetes, none of these approaches does lead to a permanent state of medicine free disease remission. Therefore, there is a need for therapies that restore physiological mechanisms of self-tolerance. Heat shock proteins (HSPs) have shown disease suppressive activities in many models of experimental autoimmune diseases through the induction of regulatory T cells (Tregs). Also in first clinical trials with HSP-based peptides in RA and diabetes, the induction of Tregs was noted. Due to their exceptionally high degree of evolutionary conservation, HSP protein sequences (peptides) are shared between the microbiota-associated bacterial species and the self-HSP in the tissues. Therefore, Treg mechanisms, such as those induced and maintained by gut mucosal tolerance for the microbiota, can play a role by targeting the more conserved HSP peptide sequences in the inflamed tissues. In addition, the stress upregulated presence of HSP in these tissues may well assist the targeting of the HSP induced Treg specifically to the sites of inflammation.

Kisspeptins (KPs) and their receptor (GPR54 or KiSS1R) play a key-role in regulation of the hypothalamic-pituitary-gonadal axis and are therefore interesting targets for therapeutic interventions in the field of reproductive endocrinology. As dogs show a rapid and robust LH response after the administration of KP10, they can serve as a good animal model for research concerning KP signaling. The aims of the present study were to test the antagonistic properties of KP analogs p234, p271, p354, and p356 in vitro, by determining the intracellular Ca2+ response of CHEM1 cells that stably express human GPR54, and to study the in vivo effects of these peptides on basal plasma LH concentration and the KP10-induced LH response in female dogs. Exposure of the CHEM1 cells to KP-10 resulted in a clear Ca2+ response. P234, p271, p354, and p356 did not prevent or lower the KP10-induced Ca2+ response. Moreover, the in vivo studies in the dogs showed that none of these supposed antagonists lowered the basal plasma LH concentration and none of the peptides lowered the KP10-induced LH response. In conclusion, p234, p271, p354, and p356 had no antagonistic effects in vitro nor any effect on basal and kisspeptin-stimulated plasma LH concentration in female dogs.

In experimental intramammary inoculation studies, it has been observed that mastitis susceptibility is influenced, among others, by cow factors. To identify milk characteristics leading to these differences, quarter milk samples of morning and evening milk were collected and analyzed for their composition (protein, fat, lactose, urea, lactoferrin, lactoperoxidase, and β-lactoglobulin concentrations), somatic cell count, and antibodies against Staphylococcus aureus. Furthermore, in vitro growth of S. aureus and Escherichia coli in fresh quarter milk samples was determined. All measured parameters differed significantly between quarters and also between morning and evening milk with the exception of lactose levels. In addition, quantitative growth of S. aureus and E. coli was significantly different in morning milk compared with evening milk. Mixed model analysis revealed that replication of S. aureus was negatively associated with the presence of fat, S. aureus-specific IgG1 antibodies, contamination of the milk sample and morning milk. Replication of E. coli was negatively associated with fat concentrations, and positively associated with morning milk. The significant difference between morning and evening milk supports the theory that changes in milk composition influence bacterial growth. Although all determined milk components differed significantly between quarters and in time no significant association with bacterial growth could be identified with the exception of fat for both studied species and IgG1 titers for S. aureus. The negative association of fat with bacterial growth was assumed to occur due to activation of lipolysis by milk handling and can most likely be neglected for in vivo relevance. The fact that S. aureus-specific IgG1 titers were negatively associated with S. aureus growth in vitro encourages the ongoing effort to develop a vaccine against S. aureus-induced mastitis., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The associations of management parameters, herd characteristics, and individual cow factors with bovine mastitis have been subject of many studies. The present study aimed to evaluate the association between milk composition parameters, including fat, protein, lactose, urea, and specific immunoglobulin levels, at the time of experimental bacterial inoculation of the mammary gland and subsequent shedding dynamics of Staphylococcus aureus. Sixty-eight cows were experimentally infected with S. aureus and closely monitored for 3 wk. Mixed model analyses were used to determine the influence of management and herd characteristics (farm and experimental group), individual cow factors (days in milk, milk yield, and quarter position), and a challenge-related parameter (inoculation dose) in combination with either the milk components fat, protein, lactose and urea, or the S. aureus-specific antibody isotype titers at the time of bacterial inoculation, on the number of S. aureus reisolated from milk after inoculation. A positive association was observed between the milk fat percentage and the number of S. aureus reisolated from quarter milk, and a negative relationship between the S. aureus-specific IgG1 titer in milk and the number of S. aureus. These findings should be considered in the development of a vaccine against S. aureus-induced bovine mastitis., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)