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.

High-Dose Filgrastim Treatment of Nonregenerative Pancytopenia Associated With Chronic Canine Ehrlichiosis.

OBJECTIVE: To report the management and outcome of a dog with canine monocytic ehrlichiosis and nonregenerative pancytopenia, with high doses of filgrastim. CASE DESCRIPTION: An 8-year-old male, mixed-breed dog, weighing 5.6kg, presented with a 1-month history of hyporexia, adynamia, and a weight loss of approximately 1kg. The general condition of the dog was observed to be poor as follows: lethargy, tachycardia, marked pallor of the mucous membranes, petechiae on the abdomen, hepatosplenomegaly, and cervical lymphadenopathy. A complete blood count analysis revealed severe leukopenia, thrombocytopenia, and anemia. A direct immunofluorescence assay using anti-Ehrlichia canis-immunoglobin G (1:400) yielded positive result. The dog was diagnosed with nonregenerative pancytopenia associated with canine monocytic ehrlichiosis. The dog presented poor prognostic signs (neutropenia, thrombocytopenia, and severe anemia). The dog was treated with antibiotics and a short course of high-dose filgrastim (50µg/kg, SC, q 48h for 4 days) to stimulate bone marrow response, prednisone to decrease peripheral platelet destruction, and an iron supplement to compensate for the iron deficiency in the bone deposits. Although temporary side effects associated with filgrastim use, such as bone pain, bleeding, and the worsening of thrombocytopenia, were observed, the treatment improved the clinical course and the cell counts in less than a month. CLINICAL RELEVANCE: The treatment protocol used in this case might be an alternative for treating cases of severe myelosuppression. This treatment plan can substantially change the clinical course of the disease for the better, compared to conventional treatment.

Hypocobalaminaemia as a cause of bone marrow failure and pancytopenia in a cat.

CASE REPORT: A male Domestic Short-hair cat was presented for chronic weight loss, lethargy and hyporexia. Complete haematological examination revealed non-regenerative anaemia, neutropenia and thrombocytopenia, as well as Howell-Jolly bodies, anisocytosis, polychromasia and macrocytosis on blood smear evaluation. Histopathological evaluation of bone marrow biopsy disclosed hypocellularity consistent with bone marrow failure. Concurrent hypocobalaminaemia was identified and treated with parenteral cyanocobalamin supplementation. Other differential diagnoses for pancytopenia, including infectious, toxic, immune-mediated and neoplastic causes, were ruled out. CONCLUSION: The cat's erythrocyte, leucocyte and platelet counts normalised after 2 months of cyanocobalamin supplementation, suggesting that pancytopenia may be a rare manifestation of feline cobalamin deficiency.

Suspected immune-mediated neutropenia and corticosteroid responsive pancytopenia in a Portuguese water dog.

An 8-year-old spayed Portuguese water dog was presented with dysuria, lethargy, and anorexia. A profound neutropenia and pancytopenia were identified. Bone marrow aspirates revealed neutrophilic hyperplasia, a significant left shift, and toxic changes, suggesting immune-mediated destruction as a likely underlying mechanism. Immunosuppressive therapy was instituted and clinical signs improved.

Neutropénie à médiation immunitaire suspectée et pancytopénie répondant aux corticostéroïdes chez un Chien d'eau portugais. Un Chien d'eau portugais femelle stérilisée âgée de 8 ans a été présentée avec de la dysurie, de la léthargie et de l'anorexie. Une neutropénie profonde et de la pancytopénie ont été identifiées. Des aspirations de la moelle osseuse ont révélé une hyperplasie neutrophilique, un virage important vers la gauche et des changements toxiques suggérant une destruction à médiation immunitaire comme mécanisme sous-jacent probable. Une thérapie immunosuppressive a été instituée et les signes cliniques se sont améliorés.(Traduit par Isabelle Vallières).

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.

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.

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.

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.

[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.

[Bovine neonatal pancytopenia in German Holstein calves]. / Bovine neonatale Panzytopenie (BNP) bei Deutschen Holstein Kälbern.

Profiles of blood cell counts were evaluated for 15 calves from three different farms. These calves showed petechia in the mucous membranes and in the skin and prolonged secondary bleeding after puncture. The clinical course of the disease could be observed in eleven calves. With exception of one case, the blood cell counts indicated a severe anaemia, leukocytopenia and thrombocytopenia. Out of these 15 calves, six calves survived and the other nine calves died or had to be euthanized due to the severity of the disease. Necropsy of these nine calves revealed petechia in the skin, subcutis, muscles, in inner organs and all serous membranes. Pathohistological examination showed a depletion of the bone marrow and lymphatic tissue in eight calves. These findings confirmed the diagnosis of bovine neonatal pancytopenia (BNP) for eight of these nine calves. Bluetongue virus serotype 8 was tested negatively using PCR. Bovine virus diarrhoea virus (BVDV) was negatively tested using immunofluorescence and cell culture and salmonella species were negatively tested in seven dissected calves. A cluster of toxins was negatively tested in one of the dissected calves. All 15 calves had high antibody titres for BVDV. The BVDV-antibody titres from twelve dams with affected calves were positive in six cases and not detectable in the other six cases. In three of the six dams with not detectable BVDV-antibody titres, calves were fed with colostrum of a further dam with high BVDV-antibody titres. In the further three dams without detectable BVDV-antibody titres, we could not ascertain which colostrum has been fed to the calves. BVDV-specific antigen could not be detected in any of the samples from the calves and dams tested. Using the activity of the gamma-glutamyl-transferase, we assumed a sufficient supply with colostrum for the examined calves.The cause for the occurrence of these BNP cases was due to bone marrow depletion.The reason for the bone marrow depletion remained unclear. However, it was obvious that the BNP described here is highly likely caused by colostrum from cows with positive BVDV-antibody titres.

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.

Ingestion of colostrum from specific cows induces Bovine Neonatal Pancytopenia (BNP) in some calves.

BACKGROUND: Since 2006, cases of haemorrhagic diathesis in young calves have been observed with a much higher incidence than previously known. The syndrome, now uniformly called Bovine Neonatal Pancytopenia (BNP), is characterized by multiple (external and internal) haemorrhages, thrombocytopenia, leukocytopenia, and bone marrow depletion. Although various infectious and toxicological causes of bleeding disorders in calves have been ruled out, the aetiology of BNP remains unknown. However, field observations have led to the hypothesis that the aetiological principle may be transmitted to calves via colostrum.The objective of the present study was to verify whether ingestion of colostrum from dams of known BNP calves can elicit signs of BNP and typical haematological findings in conveniently selected neonatal calves. Six such calves received one feeding of colostrum (or a mixture of colostrum batches) from dams of known BNP calves. As controls, another six conveniently selected calves from herds which had never had a BNP case received one feeding of colostrum from their own dams. Haematological and clinical parameters were monitored. RESULTS: One of the six experimental calves never showed any haematological, clinical or pathological evidence of BNP. In the other five calves, thrombocyte and leukocyte counts dropped within a few hours following ingestion of colostrum. Of those, three calves developed clinical signs of BNP, their post-mortem examination revealed bone marrow depletion. Of the remaining two calves, a pair of mixed twins, marked thrombocytopenia and recurrent leukocytopenia was evident in one, in which only slight changes in the bone marrow were detected, while in the other thrombocyte counts dropped, but rebounded later, and no bone marrow changes were noted. Thrombocyte counts of the experimental calves were statistically significantly lower than those of the control calves at 2 hours post ingestion of colostrum and at every sampling point between 9 hours and 8 days postcolostral. Leucocyte counts of the experimental calves were statistically significantly lower than those of control calves at 2 hours post ingestion of colostrum and 3-7 days postcolostral. CONCLUSIONS: BNP can be induced in some calves by ingestion of colostrum from cows that have given birth to BNP calves.

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.