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To detect Schmallenberg virus (SBV) infections in ruminants and to perform SBV epidemiological studies a cost-effective serological test is required. For these purposes an indirect whole virus Enzyme-linked Immunosorbent Assay (ELISA) for detection of SBV specific antibodies in ruminant blood samples was developed. Schmallenberg virus antigen was produced by propagation on Vero cells, partly purified and coated onto ELISA plates. The indirect ELISA procedure included the subsequent incubation of diluted samples, protein-G-HRP conjugate and TMB substrate solution. Net Optical Densities (OD) values were calculated and expressed as a sample to positive percentage (S/P%) by comparison of the average net OD with the OD of the positive control. Validation of this assay was performed using 633 samples from SBV-free sheep, goats and cattle, and 141 samples from SBV suspect ruminants. The diagnostic specificity was 98.8%. Test results of 86 ruminant serum samples using both the SBV-ELISA and an SBV virus neutralization test (VNT), designated as the gold standard serological test for SBV, showed good correlation: at an S/P cut-off of 15% only one VNT positive sample tested negative in the SBV ELISA. The diagnostic sensitivity of the ELISA, relative to the VNT, was 98.8% (95% CI: 93.3-100.0%). The ELISA showed a high repeatability (cv = 6.5%) and reproducibility (100% agreement). It was concluded that this ELISA is a suitable test method for the detection of SBV antibodies in sera from cows, sheep and, possibly, goats. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate the effect of vitamin E supplementation on oxidative status in blood, liver, milk, and ovarian follicular fluid in periparturient heifers. Vitamin E supplementation started 8 wk before calving and continued until 8 wk postpartum. Grass silage was the main forage fed during the experiment. In addition, supplemented heifers (n=9) received 3,000IU of vitamin E daily on a carrier food; control heifers (n=9) consumed only the carrier food. Blood samples and liver biopsies were taken frequently throughout the study and ovarian follicular fluid was sampled at 8 wk postpartum. Body condition score was scored weekly and milk yield was measured daily. A marker of oxidative damage, determinable reactive oxygen metabolites (d-ROM), and a set of antioxidants were measured in blood, liver, milk, and ovarian follicular fluid. Control heifers had a low vitamin E status, and selenium status was marginal in control and supplemented heifers. Vitamin E supplementation increased vitamin E concentrations in blood, liver, and ovarian follicular fluid and increased triacylglycerol in liver. Serum d-ROM were not reduced by vitamin E supplementation. Superoxide dismutase and glutathione peroxidase activity in red blood cells and liver and glutathione peroxidase activity in ovarian follicular fluid were not affected by vitamin E supplementation and they were not increased around calving. Protein thiol groups and ratio of reduced glutathione to oxidized glutathione were also not increased around calving. These results suggest that heifers around calving experience a low level of oxidative processes. This might be caused by lower than expected milk production attributed to a low forage intake. Serum d-ROM were negatively correlated with protein thiol groups and positively correlated with the activity of glutathione peroxidase in red blood cells, oxidized glutathione, and the ratio of reduced glutathione and oxidized glutathione in serum. The lack of treatment effects allowed estimation of the effects of body condition 4 wk before calving and the loss of body condition on markers of lipid peroxidation and antioxidants. A trend that a body condition of ⩾3 might result in more oxidative damage measured by serum d-ROM was observed, but fatter heifers had a significantly higher ratio of reduced glutathione to oxidized glutathione. [ABSTRACT FROM AUTHOR]

This study investigated the relationship between oxidative damage and the effect of vitamin E supplementation in blood, milk, and liver tissue in 16 periparturient heifers. The question is whether measurements of oxidative and vitamin E status in blood of a periparturient cow are representative of the total body, given that blood concentrations of both vitamin E and oxidative stress products change around this period. The daily vitamin E intake of the vitamin E-supplemented Holstein -Friesian heifers (n = 8) was 3,000 international units and was started 2 mo before calving; the control heifers (n = 8) were not supplemented. Oxidative damage was determined on the basis of malondialdehyde (MDA) concentrations. Blood was sampled 9 times before calving, on calving day, and twice after calving. Liver biopsies were taken at wk -5, -1, and 2 relative to calving day. Milk was obtained from all heifers immediately after calving, the first 2 milkings and on d 3, 7, and 14 at 0600 h. Serum and liver tissue were analyzed for vitamin E, cholesterol, and MDA; and milk samples were analyzed for vitamin E, MDA, fat, protein, and somatic cell count. The results showed that vitamin E supplements increased both absolute vitamin E concentrations and the ratio of vitamin E to cholesterol in blood and liver tissue. Absolute vitamin E concentration in milk tended to be greater in supplemented cows. Based on the increased MDA blood concentrations at calving, it seems that dairy heifers experience oxidative stress. The effect of vitamin E on MDA differs between the blood, liver, and mammary gland. Vitamin E supplementation could not prevent the increase in blood MDA at calving, but the significantly lower MDA blood concentrations of supplemented cows in the 2 wk after calving suggest that vitamin E has a role in recovery from parturition-related oxidative stress. Vitamin E supplementation reduced oxidative damage in liver, whereas no obvious effect was found on milk MDA concentrations. A strong relationship was found between blood and liver vitamin E and the ratio of vitamin E to cholesterol. Concentrations of MDA in blood and milk were also strongly related. The results show that the relationship between oxidative damage and vitamin E differs within blood, liver tissue, and milk. This implies that oxidative and vitamin E status calculated on the basis of blood values alone should be interpreted with caution and cannot be extrapolated to the whole animal. [ABSTRACT FROM AUTHOR]

Abstract Background At the end of 2011, a new orthobunyavirus, tentatively named Schmallenberg virus (SBV), was discovered in Germany. This virus has since been associated with clinical signs of decreased milk production, watery diarrhoea and fever in dairy cows, and subsequently also with congenital malformations in calves, lambs and goat kids. In affected countries, initial surveillance for the infection was based on examination of malformed progeny. These suspicions were followed up by real-time reverse transcription polymerase chain reaction (RT-PCR) on brain tissue. For epidemiological purposes, a serological assay was, however, needed. Results A virus neutralisation test (VNT) was developed and optimized, and subsequently evaluated. This VNT has a specificity of >99% and the sensitivity is likely also very close to 100%. The assay is highly repeatable and reproducible. The final assay was used to test for antibodies in cows, ewes and does from herds known to be infected or suspected to be so. Targets for sampling in these herds were the mothers of malformed offspring. In herds with an RT-PCR confirmed SBV infection, more than 94% (190 out of 201) of the ewes and 99% (145 out of 146) of the cows were seropositive. In herds with suspicion of SBV infection based on birth of malformed offspring only (no or negative RT-PCR), more than 90% (231 out of 255) of the ewes and 95% (795 out of 834) of the cows were seropositive. In goats, on the other hand, only a low number of seropositives was found: overall 36.4%, being 16 out of 44 goats tested. Conclusions Given the characteristics of this VNT, it can be used at a relative high throughput for testing of animals for export, surveillance, screening and research purposes, but can also be used as a confirmation test for commercially available enzyme-linked immunosorbent assays (ELISA’s) and for (relative) quantification of antibodies. Suspicions of SBV infections that were confirmed by RT-PCR were almost always confirmed by serology in cows. Due to individual registration and identification of cows and calves, affected offspring could almost always be traced back to the mother. Ewes on the other hand were not always the mothers of affected lambs, but were in many cases herd mates with unaffected lambs. This indicated a high within-herd seroprevalence of antibodies against SBV.

Background: The CD47-signal regulatory protein alpha (SIRPα) 'don't eat me' signalling axis is perhaps the most prominent innate immune checkpoint to date. However, from initial clinical trials, it is evident that monotherapy with CD47-SIRPα blocking has a limited therapeutic effect at the maximum tolerated dose. Furthermore, treatment is associated with severe side effects, most notably anaemia, that are attributable to the ubiquitous expression of CD47. Nevertheless, promising clinical responses have been reported upon combination with the tumour-targeting antibody rituximab or azacytidine, although toxicity issues still hamper clinical application., Main Body: Here, we discuss the current state of CD47-SIRPα blocking therapy with a focus on limitations of current strategies, such as depletion of red blood cells. Subsequently, we focus on innovations designed to overcome these limitations. These include novel antibody formats designed to selectively target CD47 on tumour cells as well as tumour-targeted bispecific antibodies with improved selectivity. In addition, the rationale and outcome of combinatorial approaches to improve the therapeutic effect of CD47 blockade are discussed. Such combinations include those with tumour-targeted opsonizing antibodies, systemic therapy, epigenetic drugs, other immunomodulatory T-cell-targeted therapeutics or dual immunomodulatory CD47 bispecific antibodies., Conclusion: With these advances in the design of CD47-SIRPα-targeting therapeutic strategies and increasing insight into the mechanism of action of this innate checkpoint, including the role of adaptive immunity, further advances in the clinical application of this checkpoint can be anticipated., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Objective: Unprecedented SARS-CoV-2 infections in farmed minks raised immediate concerns regarding transmission to humans and initiated intensive environmental investigations to assess occupational and environmental exposure., Methods: Air sampling was performed at infected Dutch mink farms, at farm premises and at nearby residential sites. A range of other environmental samples were collected from minks' housing units, including bedding materials. SARS-CoV-2 RNA was analysed in all samples by quantitative PCR., Results: Inside the farms, considerable levels of SARS-CoV-2 RNA were found in airborne dust, especially in personal inhalable dust samples (approximately 1000-10 000 copies/m 3 ). Most of the settling dust samples tested positive for SARS-CoV-2 RNA (82%, 75 of 92). SARS-CoV-2 RNA was not detected in outdoor air samples, except for those collected near the entrance of the most recently infected farm. Many samples of minks' housing units and surfaces contained SARS-CoV-2 RNA., Conclusions: Infected mink farms can be highly contaminated with SARS-CoV-2 RNA. This warns of occupational exposure, which was substantiated by considerable SARS-CoV-2 RNA concentrations in personal air samples. Dispersion of SARS-CoV-2 to outdoor air was found to be limited and SARS-CoV-2 RNA was not detected in air samples collected beyond farm premises, implying a negligible risk of environmental exposure to nearby communities. Our occupational and environmental risk assessment is in line with whole genome sequencing analyses showing mink-to-human transmission among farm workers, but no indications of direct zoonotic transmission events to nearby communities., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Poultry can become infected with avian influenza viruses (AIV) via (in) direct contact with infected wild birds. Free-range chicken farms in the Netherlands were shown to have a higher risk for introduction of low pathogenic avian influenza (LPAI) virus than indoor chicken farms. Therefore, during outbreaks of highly pathogenic avian influenza (HPAI), free-range layers are confined indoors as a risk mitigation measure. In this study, we characterized the seasonal patterns of AIV introductions into free-range layer farms, to determine the high-risk period. Data from the LPAI serological surveillance programme for the period 2013-2016 were used to first estimate the time of virus introduction into affected farms and then assess seasonal patterns in the risk of introduction. Time of introduction was estimated by fitting a mathematical model to seroprevalence data collected longitudinally from infected farms. For the period 2015-2016, longitudinal follow-up included monthly collections of eggs for serological testing from a cohort of 261 farms. Information on the time of introduction was then used to estimate the monthly incidence and seasonality by fitting harmonic and Poisson regression models. A significant yearly seasonal risk of introduction that lasted around 4 months (November to February) was identified with the highest risk observed in January. The risk for introduction of LPAI viruses in this period was on average four times significantly higher than the period of low risk around the summer months. Although the data for HPAI infections were limited in the period 2014-2018, a similar risk period for introduction of HPAI viruses was observed. The results of this study can be used to optimize risk-based surveillance and inform decisions on timing and duration of indoor confinement when HPAI viruses are known to circulate in the wild bird population., (© 2020 The Authors. Transboundary and Emerging Diseases published by Blackwell Verlag GmbH.)

SARS-CoV-2, the causative agent of COVID-19, caused respiratory disease outbreaks with increased mortality in 4 mink farms in the Netherlands. The most striking postmortem finding was an acute interstitial pneumonia, which was found in nearly all examined mink that died at the peak of the outbreaks. Acute alveolar damage was a consistent histopathological finding in mink that died with pneumonia. SARS-CoV-2 infections were confirmed by detection of viral RNA in throat swabs and by immunohistochemical detection of viral antigen in nasal conchae, trachea, and lung. Clinically, the outbreaks lasted for about 4 weeks but some animals were still polymerase chain reaction-positive for SARS-CoV-2 in throat swabs after clinical signs had disappeared. This is the first report of the clinical and pathological characteristics of SARS-CoV-2 outbreaks in mink farms.

Respiratory disease and increased mortality occurred in minks on two farms in the Netherlands, with interstitial pneumonia and SARS-CoV-2 RNA in organ and swab samples. On both farms, at least one worker had coronavirus disease-associated symptoms before the outbreak. Variations in mink-derived viral genomes showed between-mink transmission and no infection link between the farms. Inhalable dust contained viral RNA, indicating possible exposure of workers. One worker is assumed to have attracted the virus from mink.

No recent information is available on the specificity of current M. synoviae (Ms) and M. gallisepticum (Mg) serological tests. In this study the performance of a currently available Mg and Ms Rapid Plate Agglutination (RPA) test, and three Mg, three Ms and three Mg/Ms combination ELISAs were evaluated on SPF sera that were obtained from days (D) 0-28 after M. gallinarum , M. imitans or M. gallinaceum inoculation, after sham inoculation and without inoculation. Tracheal swabs for mycoplasma culture were obtained before inoculation (D0), 7 and 28 days post inoculation (d.p.i.) in all groups except the sham inoculated group. The different mycoplasma species colonized well. In the early stage after inoculation (7-14 d.p.i.) with heterologous mycoplasma species, the specificity varied from 85% to 100% in the Mg RPA test and from 70% to 85% in the Ms RPA test. The specificity of both Mg and Ms RPA test was 100% in the sham inoculated samples and ruled out the effect of sham medium. In the late stage (21-28 d.p.i.) specificity was 100% for both RPA tests. The test specificity was 100% for seven ELISAs except for two combination ELISAs where a specificity of 95% was found in the late stage after inoculation. However, this was not significantly different from the specificity of all other tests in the late stage of these groups. These results show that it is not advisable to establish Mg and Ms seromonitoring programmes on the Mg and Ms RPA test alone as other mycoplasma species frequently occur in poultry.

This study reports the results of diagnostic and molecular typing methods for 18 Avibacterium paragallinarum isolates obtained from outbreaks of infectious coryza in commercial layer flocks in the Netherlands. Isolation, biochemical identification, species-specific PCR tests and classical serotyping were performed. In addition, molecular typing by Enterobacterial Repetitive Intergenic Consensus-Based Polymerase Chain Reaction (ERIC-PCR) and sequence analysis of the partial HPG2 region of A. paragallinarum were applied and results of both techniques were compared. Moreover, the pathogenicity of an isolate of the most common genotype detected in the Netherlands was determined in an animal experiment. All 18 Avibacterium isolates were nicotinamide adenine dinucleotide-dependent . All isolates were detected by the species-specific conventional PCR while 33% of the isolates were missed by the species-specific real-time PCR. Sequence analysis showed a probe mismatch as a result of a single nucleotide polymorphism (G1516A). Modification of the probe of the real-time PCR was necessary to overcome false negative results. Molecular typing showed that sequence analysis of the partial HPG2 region was in concordance with ERIC-PCR results and indicated the presence of two major genotypes. Serotyping showed the presence of serovars A-1, A-2 and B-1. There was no correlation between genotyping results and serotyping results. Inoculation of an isolate of the most prevalent genotype, and belonging to serovar A-1, into brown layer hens demonstrated the pathogenicity of this isolate.

Analysis of low pathogenic avian influenza (LPAI) viruses circulating in the Netherlands in a previous study revealed associations of specific hemagglutinin (HA) and neuraminidase (NA) subtypes with wild bird or poultry hosts. In this study, we identified putative host associations in LPAI virus internal proteins. We show that LPAI viruses isolated from poultry more frequently carried the allele A variant of the nonstructural protein (NS) gene, compared to wild bird viruses. We determined the susceptibility of chickens to wild bird-associated subtypes H3N8 and H4N6 and poultry-associated subtypes H8N4 and H9N2, carrying either NS allele A or B, in an infection experiment. We observed variations in virus shedding and replication patterns, however, these did not correlate with the predicted wild bird- or poultry-associations of the viruses. The experiment demonstrated that LPAI viruses of wild bird-associated subtypes can replicate in chickens after experimental infection, despite their infrequent detection in poultry. Although the NS1 protein is known to play a role in immune modulation, no differences were detected in the limited innate immune response to LPAI virus infection. This study contributes to a better understanding of the infection dynamics of LPAI viruses in chickens., Competing Interests: The authors declare no conflict of interest.

Addition of rituximab (R) to "CHOP" (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy improved outcome for diffuse large B-cell lymphoma (DLBCL) patients. Approximately 40% of patients who receive R-CHOP still succumb to disease due to intrinsic resistance or relapse. A potential negative regulator of DLBCL treatment outcome is the CD47 "don't eat me" immune checkpoint. To delineate the impact of CD47, we used a clinically and molecularly well-annotated cohort of 939 DLBCL patients, comprising both germinal center B-cell (GCB) and non-GCB DLBCL subtypes, treated with either CHOP or R-CHOP. High (above median) CD47 mRNA expression correlated with a detrimental effect on overall survival (OS) when DLBCL patients received R-CHOP therapy ( P = 0.001), but not CHOP therapy ( P = 0.645). Accordingly, patients with low CD47 expression benefited most from the addition of rituximab to CHOP [HR, 0.32; confidence interval (CI), 0.21-0.50; P < 0.001]. This negative impact of high CD47 expression on OS after R-CHOP treatment was only evident in cancers of non-GCB origin (HR, 2.09; CI, 1.26-3.47; P = 0.004) and not in the GCB subtype (HR, 1.16; CI, 0.68-1.99; P = 0.58). This differential impact of CD47 in non-GCB and GCB was confirmed in vitro , as macrophage-mediated phagocytosis stimulated by rituximab was augmented by CD47-blocking antibody only in non-GCB cell lines. Thus, high expression of CD47 mRNA limited the benefit of addition of rituximab to CHOP in non-GCB patients, and CD47-blockade only augmented rituximab-mediated phagocytosis in non-GCB cell lines. Patients with non-GCB DLBCL may benefit from CD47-targeted therapy in addition to rituximab., (©2019 American Association for Cancer Research.)

In this study, we explore the circulation of low pathogenic avian influenza (LPAI) viruses in wild birds and poultry in the Netherlands. Surveillance data collected between 2006 and 2016 was used to evaluate subtype diversity, spatiotemporal distribution and genetic relationships between wild bird and poultry viruses. We observed close species-dependent associations among hemagglutinin and neuraminidase subtypes. Not all subtypes detected in wild birds were found in poultry, suggesting transmission to poultry is selective and likely depends on viral factors that determine host range restriction. Subtypes commonly detected in poultry were in wild birds most frequently detected in mallards and geese. Different temporal patterns in virus prevalence were observed between wild bird species. Virus detections in domestic ducks coincided with the prevalence peak in wild ducks, whereas virus detections in other poultry types were made throughout the year. Genetic analysis of the surface genes demonstrated that most poultry viruses were related to locally circulating wild bird viruses, but no direct spatiotemporal link was observed. Results indicate prolonged undetected virus circulation and frequent reassortment events with local and newly introduced viruses within the wild bird population. Increased knowledge on LPAI virus circulation can be used to improve surveillance strategies.

Poultry can become infected with low pathogenic avian influenza (LPAI) viruses via (in)direct contact with infected wild birds or by transmission of the virus between farms. This study combines routinely collected surveillance data with genetic analysis to assess the contribution of between-farm transmission to the overall incidence of LPAI virus infections in poultry. Over a 10-year surveillance period, we identified 35 potential cases of between-farm transmission in the Netherlands, of which 10 formed geographical clusters. A total of 21 LPAI viruses were isolated from nine potential between-farm transmission cases, which were further studied by genetic and epidemiological analysis. Whole genome sequence analysis identified close genetic links between infected farms in seven cases. The presence of identical deletions in the neuraminidase stalk region and minority variants provided additional indications of between-farm transmission. Spatiotemporal analysis demonstrated that genetically closely related viruses were detected within a median time interval of 8 days, and the median distance between the infected farms was significantly shorter compared to farms infected with genetically distinct viruses (6.3 versus 69.0 km; p < 0.05). The results further suggest that between-farm transmission was not restricted to holdings of the same poultry type and not related to the housing system. Although separate introductions from the wild bird reservoir cannot be excluded, our study indicates that between-farm transmission occurred in seven of nine virologically analysed cases. Based on these findings, it is likely that between-farm transmission contributes considerably to the incidence of LPAI virus infections in poultry., (© 2019 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH.)

Innate immune checkpoint CD47 has emerged as a prominent target for cancer immunotherapy and defining biomarkers predictive of response will be a crucial step towards clinical implementation. Hereto, we investigated the importance of a previously reported requisite for SLAM family member 7(SLAMF7) expression on cancer cell phagocytosis for effective CD47 antibody therapy.

CD47 is a prominent new target in cancer immunotherapy, with antagonistic antibodies currently being evaluated in clinical trials. For effective evaluation of this strategy it is crucial to identify which patients are suited for CD47-targeted therapy. In this respect, expression of the pro-phagocytic signal SLAMF7 on both macrophages and cancer cells was recently reported to be a requisite for CD47 antibody-mediated phagocytosis. Here, we demonstrate that in fact SLAMF7 expression on cancer cells is not required and does not impact on CD47 antibody therapy. Moreover, SLAMF7 also does not impact on phagocytosis induction by CD20 antibody rituximab nor associates with overall survival of Diffuse Large B-Cell Lymphoma patients. In contrast, expression of CD47 negatively impacts on overall and progression free survival. In conclusion, cancer cell expression of SLAMF7 is not required for phagocytosis and, in contrast to CD47 expression, should not be used as selection criterion for CD47-targeted therapy.

Using annual serologic surveillance data from all poultry farms in the Netherlands during 2007-2013, we quantified the risk for the introduction of low pathogenicity avian influenza virus (LPAIV) in different types of poultry production farms and putative spatial-environmental risk factors: distance from poultry farms to clay soil, waterways, and wild waterfowl areas. Outdoor-layer, turkey (meat and breeder), and duck (meat and breeder) farms had a significantly higher risk for LPAIV introduction than did indoor-layer farms. Except for outdoor-layer, all poultry types (i.e., broilers, chicken breeders, ducks, and turkeys) are kept indoors. For all production types, LPAIV risk decreased significantly with increasing distance to medium-sized waterways and with increasing distance to areas with defined wild waterfowl, but only for outdoor-layer and turkey farms. Future research should focus not only on production types but also on distance to waterways and wild bird areas. In addition, settlement of new poultry farms in high-risk areas should be discouraged.

Introduction of highly pathogenic avian influenza (HPAI) virus A(H5N8) into Europe prompted animal and human health experts to implement protective measures to prevent transmission to humans. We describe the situation in 2016 and list public health measures and recommendations in place. We summarise critical interfaces identified during the A(H5N1) and A(H5N8) outbreaks in 2014/15. Rapid exchange of information between the animal and human health sectors is critical for a timely, effective and efficient response., Competing Interests: Conflicts of Interest: None declared., (This article is copyright of ECDC, 2016.)