Transfusion von BNP-Alloantikörper-haltigem Serum auf Kälber mit unterschiedlicher Alloantigen-Reaktivität
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Beschreibung
The cause of bovine neonatal pancytopenia (BNP), which was first describes in 2006, is the uptake of colostral alloantibodies, which were formed in genetically predisposed mothers af¬ter vaccination with an inactivated BVDV vaccine (PregSure® BVD, Pfizer GmbH) that was on the market until 2010. In affected calves, the binding of these alloantibodies to platelets, leukocytes and their precursor cells in the bone marrow leads to thrombocytopenia, leukope¬nia and panmyelophthisis, which is often fatal.
In the context of this doctoral thesis, the clinical effects as well as the interindividual re¬sponse of different cell populations and other laboratory parameters to the defined transfu¬sion of serum alloantibodies from "BNP dams" should be investigated in calves in depend¬ence on the alloantibody reactivity of the leukocytes of these calves as well as on their MHC-I genotype. This should contribute to the clarification of the question to which target cells these antibodies bind in the affected calves and which haematological, clinical (haemorrhagic diathesis) and histopathological consequences (bone marrow damage) result from this. In particular, these studies also served to further verify the previously postulated pathogenesis model of a presumed interaction between the MHC-I genotype of PregSure® BVD vac¬cinated mothers and that of their calves and the occurrence of Bovine Neonatal Pancytope¬nia.
Material and Methods
In preparation for the study, serum from 22 BNP dams was stored frozen by -80°C. Serum from the eight cows with the highest alloantibody levels was finally pooled and frozen in ali-quots. For the control calves, the pooled serum of seven cows not vaccinated against BVDV was available, whose calves had proven to be haematologically inconspicuous during regular controls in the first three weeks of life.
For the actual experiment, male Holstein calves were sampled in five dairy herds between the 3rd and 5th day of life and their blood cells were tested for alloantibody reactivity in the DFZM-Allo-AK binding assay. A portion of the samples was further tested for allo-AK affinity of leukocytic MHC-I molecules. Calves included in the study were brought to the Clinic for Ruminants, University of Giessen, on day 7 of life and assigned to different groups according to their reactivity (positive or neg¬ative reaction of leukocytes to alloantibodies or MCH I positive or negative).
Results
A total of 50 calves were included in this study. Of these, data from 35 calves from the main experiment were analyzed for the present work; the dose of BNP or control serum delivered intravenously by continuous drip infusion within 24 hours was 300 ml / 50kg BW. BNP serum was given to 20 of these subjects, of which 14 animals had positive alloantibody binding re-activity to leukocytes in the DFZM-Allo-AK binding assay and 6 animals had negative alloan-tibody binding reactivity to leukocytes in the DFZM-Allo-AK binding assay. Of the 15 test calves administered control serum, 11 animals showed positive alloantibody binding reactiv¬ity against leukocytes in the DFZM-Allo-AK binding assay and 4 animals reacted negatively in this regard. In addition, the 14 calves with demonstrated positive alloantibody binding reac¬tivity against leukocytes and BNP serum to be transfused also had their MHC-I reactivity de¬termined; eight animals were found to be positive and six animals negative with respect to their allo-AK affinity for leukocyte MHC-I molecules.
None of the calves died of BNP during the course of the study, and none developed clinical signs of BNP. However, some subclinical BNP cases were diagnosed among these clinically unsuspicious calves. Nine of the 20 calves infused with BNP serum (45%) developed tempo¬rary thrombocytopenia, three (15%) developed temporary leukocytopenia, and 18 of these claves (90%) developed temporary lymphocytopenia. The differences between the two se¬rum transfusion groups were found to be significant with respect to the course of platelet counts, lymphocyte counts, and eosinophil and basophil granulocyte counts.
With regard to haematological findings, there were no detectable differences after transfusion of BNP serum and control serum, respectively, between the calves with positive and those with negative alloantibody binding reactivity, except for the number of eosinophil granulo¬cytes, which was significantly increased in the group with negative alloantibody binding reac¬tivity, and the number of monocytes, which was significantly increased in the group with posi¬tive alloantibody binding reactivity.
Similarly, no haematological differences could be detected between the calves with previ¬ously positive MHC-I binding reactivity and the calves with previously negative MHC-I binding reactivity to alloantibodies. Again, the only significant difference was in the number of eosino¬phil granulocytes.
Even administration of an significantly increased BNP serum dose from the same serum pool to three calves did not induce clinical signs of BNP in the study animals. However, they did develop subclinical BNP. Compared with the subclinical BNP cases in the main experiment, differences were observed with respect to haematologic parameters, suggesting some dose-response relationship. At the higher BNP serum dose, thrombocytopenia and lymphocytope¬nia were more pronounced and prolonged. In addition, a much more marked and strikingly prolonged decrease in leukocyte counts was noted in one of these three calves. In two calves, monocytopenia was significantly more pronounced than in the calves treated with BNP serum in the main experiment.
With regard to the substrates and proteins examined, serum enzymes, coagulation profile, and electrolytes, no significant differences were detected depending on the type of serum transfused.
When the bone marrow was examined, it was found that cytophagy of cells with subsequent decrease in bone marrow cellularity occurred significantly more frequently in the BNP serum group than in the control serum group.
Conclusions
To induce the clinical and pathological changes typical of BNP, significantly higher and/or more specific amounts of alloantibodies are obviously required than were present in the serum doses administered intravenously here. However, transfusion of the alloantibody-containing serum partially resulted in haematologic changes comparable to subclinical BNP. Thereby, the find¬ings indicated a correlation between the BNP-typical haematologic changes and the total dose of alloantibodies supplied. However, alloantibodies against bovine MHC-I are obviously not or not solely responsible for the pathogenetic effect. Therefore, the results tend to exclude a clas¬sical MHC-I variant as the causative antigene.
The different effects on the target cells circulat¬ing in the blood as well as the clear individual differences could be interpreted to the effect that, on the one hand, not all nucleated cells or their different stages of maturation express the target antigen or target antigens and that obviously no constant expression pattern of the BNP target antigens exists even within a certain cell type. It also cannot be excluded that several alloantibodies directed against different epitopes are produced to different extents by the BNP dams, which could explain the different haematological changes even in naturally occurring BNP cases.
Autor*in
Susanne Viebrock