A Functionally Different Immune Phenotype in Cattle Is Associated With Higher Mastitis Incidence.

A novel vaccine against bovine viral diarrhea (BVD) induced pathogenic antibody production in 5-10% of BVD-vaccinated cows. Transfer of these antibodies via colostrum caused Bovine neonatal pancytopenia (BNP) in calves, with a lethality rate of 90%. The exact immunological mechanisms behind the onset of BNP are not fully understood to date. To gain further insight into these mechanisms, we analyzed the immune proteome from alloreactive antibody producers (BNP cows) and non-responders. After in vitro stimulation of peripheral blood derived lymphocytes (PBL), we detected distinctly deviant expression levels of several master regulators of immune responses in BNP cells, pointing to a changed immune phenotype with severe dysregulation of immune response in BNP cows. Interestingly, we also found this response pattern in 22% of non-BVD-vaccinated cows, indicating a genetic predisposition of this immune deviant (ID) phenotype in cattle. We additionally analyzed the functional correlation of the ID phenotype with 10 health parameters and 6 diseases in a retrospective study over 38 months. The significantly increased prevalence of mastitis among ID cows emphasizes the clinical relevance of this deviant immune response and its potential impact on the ability to fight infections.

Bovine Neonatal Pancytopenia is a heritable trait of the dam rather than the calf and correlates with the magnitude of vaccine induced maternal alloantibodies not the MHC haplotype.

Bovine Neonatal Pancytopenia (BNP), a bleeding syndrome of neonatal calves, is caused by alloantibodies absorbed from the colostrum of particular cows. A commercial BVD vaccine is the likely source of alloantigens eliciting BNP associated alloantibodies. We hypothesized that the rare occurrence of BNP in calves born to vaccinated dams could be associated with genetic differences within dams and calves. We found that the development of BNP within calves was a heritable trait for dams, not for calves and had a high heritability of 19%. To elucidate which genes play a role in the development of BNP we sequenced candidate genes and characterized BNP alloantibodies. Alloantigens present in the vaccine have to be presented to the dam's immune system via MHC class II, however sequencing of DRB3 showed no differences in MHC class II haplotype between BNP and non-BNP dams. MHC class I, a highly polymorphic alloantigen, is an important target of BNP alloantibodies. Using a novel sequence based MHC class I typing method, we found no association of BNP with MHC class I haplotype distribution in dams or calves. Alloantibodies were detected in both vaccinated BNP and non-BNP dams and we found no differences in alloantibody characteristics between these groups, but alloantibody levels were significantly higher in BNP dams. We concluded that the development of BNP in calves is a heritable trait of the dam rather than the calf and genetic differences between BNP and non-BNP dams are likely due to genes controlling the quantitative alloantibody response following vaccination.

Colostrum from cows immunized with a vaccine associated with bovine neonatal pancytopenia contains allo-antibodies that cross-react with human MHC-I molecules.

In 2006, a new haemorrhagic syndrome affecting newborn calves, Bovine Neonatal Pancytopenia (BNP), was reported in southern Germany. It is characterized by severe bleeding, destruction of the red bone marrow, and a high case fatality rate. The syndrome is caused by alloreactive, maternal antibodies that are ingested by the calf with colostrum and result from a dam vaccination with one particular vaccine against Bovine-Viral-Diarrhoea-Virus. Because bovine colostrum is increasingly gaining interest as a dietary supplement for human consumption, the current study was initiated to elucidate whether BNP alloantibodies from BNP dams (i.e. animals that gave birth to a BNP-affected calf) cross-react with human cells, which could pose a health hazard for human consumers of colostral products. The present study clearly demonstrates that BNP alloantibodies cross-react with human lymphocytes in vitro. In agreement with previous reports on BNP, the cross-reactive antibodies are specific for MHC-I molecules, and sensitize opsonised human cells for in vitro complement lysis. Cross-reactive antibodies are present in serum and colostrum of individual BNP dams. They can be traced in commercial colostrum powder manufactured from cows immunized with the vaccine associated with BNP, but are absent from commercial powder manufactured from colostrum excluding such vaccinated cows. In humans alloreactive, MHC-I specific antibodies are generally not believed to cause severe symptoms. However, to minimize any theoretical risk for human consumers, manufacturers of bovine colostrum for human consumption should consider using only colostrum from animals that have not been exposed to the vaccine associated with BNP.

Immune mechanism and clinical significance of macrophage to medullary hematopoietic injury of immune-related hematocytopenia patients.

BACKGROUND: Immune-related hematocytopenia (IRH) is considered to be related with the production of autoantibody, as well as the activation of humoral immunity which is stimulated by B lymphocyte. This study aimed to observe the levels of various cytokines in the blood serum and the in situ active state of macrophage (Mφ) in the medullary hematopoietic microenvironment of IRH patients, and to probe into the immune mechanism and clinical significance of Mφ in hematopoietic cell injury. METHODS: ELISA is used to detect the IL-4, IL-6, IL-12, IL-17, and IFN-γ levels in the peripheral blood serum of 376 patients in pre- and post-therapy. Cytochemistry and cell immunochemistry methods are used to observe the peroxidase (POX), nonspecific esterase (NSE), hemosiderin granules, and HLA-DR activity of Mφ in the bone marrow of patients. Immunofluorescence is used to observe the expression of hemocyte antihuman globulin IgG antibody, lymphocytes CD4 molecule, Mφ membrane FcγIIreceptor (FcγIIR), mannitose receptor (MR), IFN-γ, ICAM-1, IL-12, and IL-17A and the formation mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC) hematopoietic cell islands (HI) in the medullary hematopoietic microenvironment of patients. Glucocorticoid is used for treatment on the basis of anti-infection therapy, and gamma globulin stoss therapy is used for the appearance of ADCC-type HI or serious Mφ bloodthirsty phenomenon; if necessary, association of Cyclosporine A (CsA) should be used and chalybeate should be supplemented. RESULTS: In the patient group, the levels of IL-4, IL-6, IL-12, IL-17, and IFN-γ were increased. After treatment, the cytokine levels gradually became normal. The activated Mφ in the marrow highly expressed NSE and POX, and Mφ swallowed more hemosiderin particles, but the iron in the cytoplasm of immature erythrocytes decreased. The activated Mφ expressed HLA-DR, MR, ICAM-1, IFN-γ, and IL-12. For patients with humoral immunity activation and bacterial infection, Mφ weakly expressed IL-17A but highly expressed FcγIIR, and the phenomenon that ADCC-type HI broke pathological blood corpuscles often occurred; for the cellular immune activation along with virus infection, the white blood count (WBC) significantly reduced, Mφ weakly expressed FcγIIR, secretory highly expressed IL-17A, and the phenomena that Mφ adhered to, captured and swallowed blood cell often occurred. After four weeks of anti-infective and immunosuppressive therapy, nuclear apoptosis of Mφ occurred in the bone marrow of patients, HI and bloodthirsty phenomenon disappeared, and the peripheral blood picture started to improve. CONCLUSIONS: Mφ is an important antigen presenting cell in the IRH marrow for hematopoiesis destruction and an immune effector cell of hematopoietic injury; infection can promote the activation of Mφ, upregulate the impression of immune molecule and receptors, form ADCC HI, aggravate hematopoietic injury, and accelerate the destruction on hematopoietic cell.

Reproduction of bovine neonatal pancytopenia (BNP) by feeding pooled colostrum reveals variable alloantibody damage to different haematopoietic lineages.

Bovine neonatal pancytopenia (BNP) is a recently described haemorrhagic disease of calves characterised by thrombocytopenia, leucopenia and bone marrow depletion. Feeding colostrum from cows that have previously produced a BNP affected calf has been shown to induce the disease in some calves, leading to the hypothesis that alloantibodies in colostrum from dams of affected calves mediate destruction of blood and bone marrow cells in the recipient calves. The aims of the current experimental study were first to confirm the role of colostrum-derived antibody in mediating the disease and second to investigate the haematopoietic cell lineages and maturation stages depleted by the causative antibodies. Clinical, haematological and pathological changes were examined in 5 calves given a standardised pool of colostrum from known BNP dams, and 5 control calves given an equivalent pool of colostrum from non-BNP dams. All calves fed challenge colostrum showed progressive depletion of bone marrow haematopoietic cells and haematological changes consistent with the development of BNP. Administration of a standardised dose of the same colostrum pool to each calf resulted in a consistent response within the groups, allowing detailed interpretation of the cellular changes not previously described. Analyses of blood and serial bone marrow changes revealed evidence of differential effects on different blood cell lineages. Peripheral blood cell depletion was confined to leucocytes and platelets, while bone marrow damage occurred to the primitive precursors and lineage committed cells of the thrombocyte, lymphocyte and monocyte lineages, but only to the more primitive precursors in the neutrophil, erythrocyte and eosinophil lineages. Such differences between lineages may reflect cell type-dependent differences in levels of expression or conformational nature of the target antigens.

A colostrum substitute prevents bovine neonatal pancytopenia (BNP) in a herd with previously BNP-affected calves.

The objective of this study was to demonstrate that bovine neonatal pancytopenia (BNP) can be prevented when intake of maternal colostrum is prevented in a dairy farm with verified BNP cases. A group of 30 female calves was fed with a colostrum substitute instead of maternal colostrum (group A) whereas the control group of 30 female calves was fed with the colostrum of their own mothers (group B). The female calves were randomly assigned to groups A or B. All 60 calves were daily blood sampled in the first eleven days of life, afterwards up to the age of three weeks one blood sample was taken every other day. All blood samples were analyzed for thrombocyte and leucocyte counts. In addition, 113 calves of both sexes, which were born during the trial period, were blood sampled once at 6-10 days old. In group A, no BNP positive calf was verified. In group B, eight calves with a significant decrease of thrombocyte and leucocyte counts were observed. Only one of these eight calves had clinical signs consistent with BNP and the other seven calves were classified as subclinical BNP cases. Of the other 113 contemporary calves, eleven animals had clinical signs of BNP accompanied by a decrease of thrombocyte and leucocyte counts and four of these eleven calves died due to BNP. Our results revealed that replacement of maternal colostrum can prevent subclinical and clinical cases of BNP as well as losses due to BNP in a dairy herd with verified BNP-cases and in addition, that colostrum from these cows was the major risk factor for BNP in this dairy herd.

Demonstration of early functional compromise of bone marrow derived hematopoietic progenitor cells during bovine neonatal pancytopenia through in vitro culture of bone marrow biopsies.

BACKGROUND: Bovine neonatal pancytopenia (BNP) is a syndrome characterised by thrombocytopenia associated with marked bone marrow destruction in calves, widely reported since 2007 in several European countries and since 2011 in New Zealand. The disease is epidemiologically associated with the use of an inactivated bovine virus diarrhoea (BVD) vaccine and is currently considered to be caused by absorption of colostral antibody produced by some vaccinated cows ("BNP dams"). Alloantibodies capable of binding to the leukocyte surface have been detected in BNP dams and antibodies recognising bovine MHC class I and ß-2-microglobulin have been detected in vaccinated cattle. In this study, calves were challenged with pooled colostrum collected from BNP dams or from non-BNP dams and their bone marrow hematopoietic progenitor cells (HPC) cultured in vitro from sternal biopsies taken at 24 hours and 6 days post-challenge. RESULTS: Clonogenic assay demonstrated that CFU-GEMM (colony forming unit-granulocyte/erythroid/macrophage/megakaryocyte; pluripotential progenitor cell) colony development was compromised from HPCs harvested as early as 24 hour post-challenge. By 6 days post challenge, HPCs harvested from challenged calves failed to develop CFU-E (erythroid) colonies and the development of both CFU-GEMM and CFU-GM (granulocyte/macrophage) was markedly reduced. CONCLUSION: This study suggests that the bone marrow pathology and clinical signs associated with BNP are related to an insult which compromises the pluripotential progenitor cell within the first 24 hours of life but that this does not initially include all cell types.

Immunophenotyping and characterization of BNP colostra revealed pathogenic alloantibodies of IgG1 subclass with specifity to platelets, granulocytes and monocytes of all maturation stages.

Bovine neonatal pancytopenia (BNP) is mainly characterized by multiple haemorrhages, thrombocytopenia and leukocytopenia as a result of bone marrow depletion. BNP can be induced in healthy calves through application of colostrum from BNP donors, proofing that BNP is mediated to maternal alloantibodies. Alloantibody binding to bovine blood cells is present in sera and colostra of BNP donors and is probably initialized by vaccination with a certain BVD vaccine. To understand etiology and pathomechanisms of BNP, we closely characterized disease inducing antibodies regarding immunoglobulin subclass and binding specificities to peripheral blood derived leukocytes and platelets. By exact phenotyping the targeted blood cell subsets, including platelets for the first time, we investigated that BNP alloantibodies are exclusively of IgG1 subclass. Interestingly, IgG1 of BNP colostra bound to 70% leukocytes and 100% platelets irrespective of different bovine breeds and cellular maturity of all specimens tested. Furthermore, staining pattern on platelets as well as leukocyte subsets by BNP-IgG1 alloantibody exposed 100% reactivity to platelets, granulocytes and monocytes. Interestingly, the main part of T-helper cells was not bound by colostral alloantibodies. Our results point to a crucial role of IgG1 antibodies in BNP and to a target antigen that is expressed by all cells of myeloid lineage, but only partially by the lymphoid lineage.

[Reproducibility of bovine neonatal pancytopenia (BNP) via the application of colostrum]. / Reproduzierbarkeit der bovinen neonatalen Panzytopenie (BNP) über die Verabreichung von Kolostrum.

A haemorrhagic diathesis has been observed in young calves since 2007 which is described as bovine neonatal pancytopenia (BNP) and presents a completely new disease. The objectives of our investigation were to test if BNP could be reproduced using colostrum of cows with a BNP history and pre-colostral calves from farms where BNP has not been observed. In the present experiment, 22 German Holstein calves from BNP-free farms were fed four to six hours after birth 2.5 l colostrum from cows which had been reported to have had at least one calf with BNP in the last lactation. We distinguished three different experimental groups according to the composition of the colostrum. In experimental group I, each of the six calves received colostrum of a single cow, in experimental group II all six calves received colostrum from the same cow and in experimental group III each of the ten calves received a colostrum mix from ten different cows. Clinical signs of BNP were observed in 50% of the calves in experimental group I, 67% of the calves in experimental group II and all calves in experimental group III. The lethality in the three experimental groups was significantly different with rates of 16.7%, 66.7% and 80%, respectively. Calves fed with a colostrum-mix in experimental group III had the highest lethality. Neither the farm nor the amount of the colostrum fed had a significant effect on the occurrence and course of BNP. The profiles for thrombocytes, leucocytes and erythrocytes significantly differed in dependence of the severity of BNP signs. Calves with non-lethal BNP showed thrombocytopenia with values below 100 G/l on the 1th to 3rd and the 7th to 11th day of life. In calves with lethal BNP, thrombocytes decreased under 50 G/l from day 5. In calves with non-lethal BNP, a decrease of the leucocytes under the threshold was present only for a short period of time. In calves with lethal BNP, leucocytes decreased in the first 5 days after birth continuously and increased on the 6th to the 8th day to normal values and then a rapid decrease occurred. Erythrocytes decreased under the normal threshold just in the last two days before the calves died or were euthanized. Thus, the present experiments showed that colostrum of cows with a BNP-history and vaccination with PregSure BVD from Pfizer caused lethal BNP. We can assume that the different reactions of the calves are due to immunogenetic reactions to colostral alloreactive antibodies. The reaction spectrum of calves depends on the presence of antigens which can react with these colostral antibodies. The experimental results can explain the different incidences of BNP within and among farms as well as between breeds.

Vaccine-induced antibodies linked to bovine neonatal pancytopenia (BNP) recognize cattle major histocompatibility complex class I (MHC I).

A mysterious disease affecting calves, named bovine neonatal pancytopenia (BNP), emerged in 2007 in several European countries. Epidemiological studies revealed a connection between BNP and vaccination with an inactivated vaccine against bovine virus diarrhea (BVD). Alloantibodies reacting with blood leukocytes of calves were detected in serum and colostrum of dams, which have given birth to calves affected by BNP. To understand the linkage between vaccination and the development of alloantibodies, we determined the antigens reacting with these alloantibodies. Immunoprecipitation of surface proteins from bovine leukocytes and kidney cells using sera from dams with a confirmed case of BNP in their gestation history reacted with two dominant protein species of 44 and 12 kDa. These proteins were not detected by sera from dams, free of BVDV and not vaccinated against BVD, and from sera of animals vaccinated with a different inactivated BVD vaccine. The 44 kDa protein was identified by mass spectrometry analysis as MHC I, the other as ß-2-microglobulin. The presence of major histocompatibility complex class I (MHC I) in the vaccine was confirmed by Western blot using a MHC I specific monoclonal antibody. A model of BNP pathogenesis is proposed.

Bovine Neonatal Pancytopenia: is this alloimmune syndrome caused by vaccine-induced alloreactive antibodies?

Bovine Neonatal Pancytopenia (BNP) is a new emerging disease observed since 2007 in Germany and neighbouring countries. The syndrome affects newborn calves and is characterized by pancytopenia, severe bleeding and high lethality. So far, a causative role of infectious or toxic agents has been ruled out. Instead, the syndrome is induced after ingestion of colostrum, the first milk that supplies the calf with maternal antibodies. In analogy to similar diseases in humans it has therefore been postulated that BNP is caused by alloreactive, maternal antibodies. There is a striking association between BNP and a previous vaccination of the respective dams with a particular vaccine against Bovine Virus Diarrhoea (BVD). This association has led to a suspension of the marketing authorisation for the vaccine, by the European Commission. The current study investigates the role of this vaccine in the pathogenesis of BNP. By flow cytometry we were able to demonstrate that sera of BNP dams (dams that gave birth to a BNP calf) harbour alloreactive antibodies binding to surface antigens on bovine leukocytes. A significantly weaker alloreactivity was observed with sera of non-BNP dams that have been vaccinated with the same vaccine but delivered healthy calves. No binding was seen with non-BVD-vaccinated control cows and animals that were vaccinated with other inactivated BVD vaccines so far not associated with BNP. The binding is functionally relevant, because opsonization of bovine leukocytes with alloantibodies led to an elevated cytophagocytosis by bovine macrophages. To test whether the vaccine induces alloreactive antibodies two strategies were employed: Guinea pigs were vaccinated with a panel of commercially available BVD-vaccines. Only the incriminated vaccine induced antibodies binding surface antigens on bovine leukocytes. Additionally, two calves were repeatedly vaccinated with the suspected vaccine and the development of alloreactivity was monitored. In dependence of the number of booster immunizations the induction of alloreactive antibodies could be observed. Finally, by affinity purification we were able to directly demonstrate that BNP associated alloantibodies cross react with the bovine kidney cell line used for vaccine production. Together this provides strong evidence that this particular BVD vaccine has the potential to induce BNP associated alloantibodies.

Detection of colostrum-derived alloantibodies in calves with bovine neonatal pancytopenia.

Bovine neonatal pancytopenia (BNP) is an emerging calf disease of unknown cause characterized by a pronounced susceptibility to bleeding as a result of a pancytopenia and bone marrow depletion. In this study we investigated whether this phenomenon is related to colostrum-derived alloantibodies directed against neonatal leukocytes. In a first experiment and using a flow cytometric approach sera from 6 BNP-dams (had given birth to BNP-calves; vaccinated against bovine viral diarrhea virus [BVDV]) and 6 control-dams (no herd history of BNP; no BVDV vaccination) were analyzed for the presences of alloantibodies (IgG) able to bind to the surface of leukocytes isolated from 7 calves from a herd with no history of BNP (no BVDV vaccination). In a second experiment, 4 neonates from 3 BNP-dams were fed colostrum from their corresponding mothers and sampled on a regular basis from birth up to day 21 of life under clinically controlled conditions. Sample analysis of the 4 neonates included hematology (white blood cell count and platelets), bone marrow cytology and histopathology as well as the flow cytometric detection of the percentage of IgG+-lymphocytes/monocytes in the peripheral blood. Experiment #1 showed that all BNP-dam sera harbored significantly higher alloantibody titers than the control dam sera (p<0.001). In the peripheral blood of the two neonates (Experiment #2), the percentage of IgG+-cells increased dramatically within 12h post colostrum intake (p.c.i.), remaining at over 95% for up to 3 days. Both calves developed BNP-associated clinical symptoms, one died. Both twin calves showed no clinical symptoms accompanied by a minor increase of IgG+ cells for up to 12h. Thus, the level of IgG+-cells and the duration of the detection thereof correlated with the severity of BNP developed by these animals. The results show that BNP-dams harbor alloantibodies against surface antigens of neonatal leukocytes in their sera that are readily transferred to the offspring via colostrum. These alloantibodies probably play a crucial role in the pathogenesis of BNP.