Bovine neonatal pancytopenia and beyond
Sandra F.E. Scholes BVM&S PhD MRCVS FRCPath DipECVP
Animal Health and Veterinary Laboratories Agency Lasswade, Pentlands Science Park, Bush Loan,
Penicuik, EH26 0PZ, Scotland, UK.
Background
A bleeding disorder of cattle of undetermined cause observed in southern Germany in 2006 1 has
since then been reported in cattle in many countries across Europe, with the exception of countries
without vaccination against BVDV, and more recently in New Zealand2 3 4 5. By the end of 2010, more
than 4000 cases had been reported in the EU5 and cases continue to be reported6. The clinical
presentation and pathology are now well recognised as a bleeding disorder of calves less than four
weeks old, occurring independent of breed or sex, with a high level of mortality2 7; the underlying bone
marrow pathology is trilineage hypoplasia, defined as concurrent depletion of erythroid and myeloid
cells and megakaryocytes resulting in less than 25% cellularity8 9. The bone marrow lesion, which
has been referred to as panmyelophthisis or bone marrow aplasia2 5, results in thrombocytopenia,
leukopenia (neutropenia and lymphopenia) and a non-regenerative anemia10, the latter varying in
severity in relation to the degree of hemorrhage. The condition was designated Bovine Neonatal
Pancytopenia (BNP) at the Satellite Symposium of the European Buiatric Congress in 2009 5 11; the
generally accepted diagnostic criteria are multiple hemorrhages and bone marrow trilineage
hypoplasia in a calf younger than 4 weeks of age3 9. The incidence of clinical BNP on most affected
farms is low, but the case fatality rate is very high and can become important at the individual farm
level, with losses of up to 5% of calves in a herd being reported7 10. It is worthy of note that a very
similar or identical clinical and pathological presentation had occurred previously, for example in a calf
in Canada12, and in two calves in Germany in 1989 and 1991, the latter noted from a retrospective
analysis of cases submitted to the Clinic for Ruminants, LMU, Munich, Germany during the 20 years
prior to 20063.
An initial study reported the detection of circovirus DNA in 5 out of 25 cases of BNP but also in 1 out
of 8 unaffected control calves13; however further studies have found no evidence to suggest a viral
etiology for BNP7 14. Farm investigations and toxicological analyses did not detect evidence of toxins
known to induce bone marrow suppression2. Limited studies have been unable to demonstrate a
simple mode of inheritance by investigation of a mutation in factor XI15 or by investigation of major
histocompatibility complex allelic frequencies16. While no linkage to MHC-class II or factor XI has
been detected in affected calves there is some evidence that within herds, specific groups or lines of
animals may be more frequently affected, suggesting that some animals are more at risk of BNP,
possibly due either to intrinsic factors or to interaction between the pregnant cow and its fetus 15 16.
Case control studies have provided strong evidence for an association between BNP and the use of a
Bovine Viral Diarrhoea Virus (BVDV) vaccine (PregSure(®)BVD; Pfizer Animal Health)9 17, in
concordance with early field observations. PregSure(®)BVD was removed voluntarily from the
European market in 201018. This inactivated vaccine incorporated a novel adjuvant and was shown to
produce a significantly higher virus neutralising antibody titre than other commercially available
inactivated BVDV vaccines containing different adjuvants 5. Nevertheless, the overall incidence of
clinical BNP appears low in relation to the number of doses of PregSure® BVD sold across a number
of European countries. The overall incidence for BNP at European Union level between 2004 and
2009 was estimated to be 0.016% based on a single dose of PregSure® BVD18. Consequently, it is
possible that other factors play an important role in the aetiology of BNP. The incidence of BNP
varied widely between different regions within countries: for example comparison of the BNP rates in
the German Federal States of Bavaria and Lower Saxony revealed 100 cases per 100,000 doses
PregSure(®)BVD in the former and as few as 6 cases per 100,000 doses in the latter19. In Bavaria
PregSure(®)BVD was used according to the instructions for use whereas in Lower Saxony, BVDVimmunization was performed according to a two-step vaccination protocol including a first
1
immunization with an inactivated BVDV-vaccine followed by booster immunizations with a liveattenuated BVDV-vaccine. As a consequence, those cattle that received PregSure(®)BVD received in
general more than two doses in Bavaria, while in Lower Saxony cows received a maximum of one
dose19.
Aspects of pathogenesis
Animal studies
The clinicopathologic features of BNP can be reproduced by feeding of colostrum (‘BNP colostrum’)
from cows which have borne affected calves (‘BNP dams’) to unrelated recipient calves 3 20 21 22.
Disease can be prevented by colostrum substitution 23. These results support the hypothesis that
colostrum from dams of affected calves contains immunoglobulins that can mediate destruction of
blood and bone marrow cells in the recipient calves via an alloimmune reaction.
While feeding individual BNP colostrum has a more variable outcome in terms of development of
BNP3 20, the use of pooled colostrum from a number of BNP dams produces a more reliable outcome
with a high proportion (80-100%) of calves developing BNP or its typical clinicopathological features 20
21 22. Analyses of blood and serial bone marrow changes in 7 calves given a standardised pool of BNP
colostrum compared with 7 control calves given an equivalent pool of colostrum from non-BNP dams
revealed progressive depletion of bone marrow haematopoietic cells and haematological changes
consistent with the development of BNP in the BNP colostrum group21 22. Sternal bone marrow from
BNP-colostrum challenged calves and the control calves had moderately abundant myeloid and
erythroid series cells and megakaryocytes at varying stages of maturation at day 1, whereas in the
challenge calves at day 6 there were marked reductions in the numbers of megakaryocytes, which
were mostly mature, and in myeloid and erythroid series cells (mainly comprising mature neutrophils
and eosinophils and late normoblasts). Marked bone marrow depletion was observed in all 7 calves at
day 10 post BNP colostrum administration21 22. As early as 4 hours following challenge colostrum
ingestion the numbers of lymphocytes, neutrophils and monocytes had dropped by 75-95% compared
to controls and by 8 hours the numbers of thrombocytes were approximately 40% of control calves’
values. Such rapid depletion of peripheral blood cells suggests a pathogenesis involving complementmediated lysis and/or enhanced phagocytosis of cells, mediated by the alloantibody22. Lymphopenia
has been observed post-BNP colostral challenge in several studies3 20 21 22 24. Investigations to
determine whether a specific subset of peripheral blood mononuclear cells was depleted failed to
demonstrate any subset-specific depletion, but did observe a tendency for CD25+ve, γδT cell and B
cell percentages to drop and subsequently fail to rise when compared to control animals21.
Bone marrow hematopoietic progenitor cell cultures
A pilot study using bone marrow hematopoietic progenitor (BM-HPC) cultures assessed the
functionality of bovine BM-HPCs harvested from calves 24 hours and 6 days post challenge with
either pooled BNP colostrum or pooled normal colostrum 21. 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 indicating functional compromise of the HPCs prior to the development of clinical signs
and histopathological lesions. 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, thus indicating that the main target cell is the
pluripotent HPC as the more differentiated cells (CFU-E and CFU-GM precursors) were apparently
not compromised at 24 hours post-colostrum ingestion. 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
types21.
Alloantibody response – possible targets
Antibodies present in serum of BNP dams have been shown to be capable of binding to peripheral
leukocytes and to cells in bone marrow smears of unrelated normal calves 24 and were detected bound
to leukocytes of affected calves5 25. Such studies support the hypothesis that maternally derived
colostral alloantibody targeting a bovine antigen (or antigens) causes the bone marrow pathology in
2
calves, although their exact role in the pathogenesis of the lesions and the identity of the target
antigen(s) is as yet unconfirmed. Antibodies in serum from BNP-dams have been shown to react with
the bovine kidney cell line used in PregSure(®)BVD manufacture5 and to recognise bovine MHC class
I (MHC I) proteins11 26, suggesting that the stimulus for alloantibody production is cell culture
components within the vaccine.
More recently, Euler and co-workers1 characterized and compared the proteome of the BNPassociated vaccine and another vaccine directed against BVDV but not related to BNP, and the cell
surface proteome of MDBK (Madin-Darby Bovine Kidney) cells, the cell line used for production of the
BNP-associated vaccine. The number of proteins identified in the BNP related vaccine exceeded the
amount of proteins identified in the non-BNP related vaccine and was almost as high as the number
of surface proteins detected on MDBK cells; the large number of shared cellular and serum derived
proteins indicated that the BNP associated vaccine contained many molecules originating from MDBK
cells and vaccine production. Within the proteins detected in both the BNP related vaccine and on
MDBK cell surface, further possible BNP candidate alloantigens were identified including vitamin D
binding protein, thrombospondin-1, alpha-1-acid-glycoprotein, alpha-1-antiproteinase, and adenylate
kinase 2 (AK 2), with the latter in particular being suggested as worthy of further analysis.
Discussion
The evidence of very early removal of lymphocytes from the peripheral circulation, taken in
association with the demonstration of binding of alloantibodies11 25 26 and the results of bone marrow
hematopoietic progenitor cell (HPC) cultures21 support the hypothesis that one or more common
antigen(s) present on bone marrow HPCs and lymphocytes are targeted almost immediately after
colostrum absorption.
The relative consistency of the clinicopathological findings in calves challenged with pooled BNP
colostrum20 21 contrasts with experimental findings that individual calves varied in their susceptibility
following feeding of individual BNP colostrum 3 20. This indicates that pooling of alloantibodies from
different cows reduced this variation, suggesting that the alloantibody specificities varied between
individual cows and that pooling increased the likelihood of antibodies reactive with most calves. The
slight variation that remained did not correlate with serum IgG concentrations in individual challenge
calves, consistent with the idea that specific alloantibodies within the colostrum, rather than IgG
concentrations per se, are responsible for BNP pathology22. These findings suggest that the colostral
alloantibodies responsible for mediating the disease are directed against a highly polymorphic set of
antigens.
As in other species, cattle express several MHC I genes that are highly polymorphic 27. The hypothesis
that the alloantibodies that cause BNP are stimulated by cell culture components within the vaccine
and are directed against MHC-15 11 26 would restrict the occurrence of BNP to calves that share
common MHC I epitopes with the vaccine cell-line and originate from dams that do not share some or
all of these epitopes. This hypothesis is consistent with observations by Krappmann and others15 who
found a significant accumulation of BNP cases within a specific pedigree, suggesting a familial
component to the development of BNP. While the induction of such antibodies during BNP has been
demonstrated, it is possible that specificity for the broadly expressed classical MHC-I molecules may
not be responsible for the disease syndrome as they might be expected to target cells more widely
than the haematopoietic lineages (bone marrow and peripheral lymphocytes). Expression of MHC-I is
widespread and includes both CFU-E and CFU-GM in cattle28 29 however there is an apparent sparing
effect on CFU-E and CFU-GM at 24 hours post BNP colostrum administration21; the presence of
MHC-I on bovine CFU-GEMM, whilst expected, has not been confirmed. Possible explanations could
include a difference in the levels of MHC-I expression in the highly active CFU-GEMM, a difference in
their susceptibility to damage, or a difference in cell-type specific expression of an unusual nonclassical MHC-I specificity which could make these cells more sensitive to antibody-dependent
damage (either via antibody-dependent cell-mediated cytotoxicity or complement-mediated)21.
Further candidate alloantibody targets have been identified, for example AK2 1. Mutations in the AK2
gene may result in monocytopenia, neutropenia, and lymphopenia combined with normal erythrocyte
3
count and thrombocytopenia in some cases30 however loss of AK 2 does not interfere with
development of immature hematopoietic cells or erythropoiesis, but with lymphocyte development31.
The production of vaccines using antigen produced in same-species cell lines is not uncommon, and
yet disease associated with alloantibody reaction against nucleated cells has not been reported prior
to the emergence of BNP, suggesting that another component of PregSure(®)BVD, for example the
adjuvant, may be important in the development of BNP 22. It is possible that the vaccine adjuvant
amplifies not only the production of alloantibody against components of the vaccine but also boosts
normal pregnancy-induced maternal alloantibodies against paternally-derived fetal MHC antigens.
The latter have previously been detected in sera of healthy cows during or following pregnancy 32 33.
Although usually of low titre and generally assumed to be non-pathogenic, it is possible to speculate
that the presence of high levels of such alloantibodies may have been responsible for the BNP-like
cases with no history of PregSure(®)BVD association3 12 34 35 36. A comparison of the pathogenesis of
vaccination associated and sporadic cases of BNP may be informative, particularly as alloimmune
pancytopenia characterised by trilineage hypoplasia does not appear to have been confirmed in
species other than cattle. The continuing incidence of BNP following withdrawal of the vaccine6 may
suggest that boosting of the cow’s alloantibody titer by subsequent pregnancies may contribute to the
disease and supports this hypothesis further, indicating that the role of fetus-induced alloantibodies in
BNP warrants further investigation22. The opportunities for systematic investigation of the
pathogenesis of BNP has been limited by the lack of an animal model, reliance on clinical cases and
the requirement to use colostral challenge of calves. The use of BM-HPC cultures will facilitate in
vitro studies to further characterise the aetiology, maternal vaccinal responses, colostral antibody titre
and specificity in a standardised, non-animal model system21.
These field and experimental investigations of BNP demonstrate the pivotal role of pathology in
identifying and characterising emerging disease threats, epidemiological investigations and analysis
of the pathogenesis. Although there are many definitions of surveillance, all incorporate the following
main elements: ‘The ongoing collection, validation, analysis and interpretation of health and disease
data that are needed to inform key stakeholders in order to permit them to take action by planning and
implementing more effective, evidence based public health policies and strategies relevant to the
prevention and control of disease or disease outbreaks. The prompt dissemination of information to
those who need to know is as essential as ensuring the quality, validity and comparability of the
data’37. Analysis of laboratory data continues to offer the greatest potential for syndromic
surveillance in veterinary medicine38. Whilst the important role of veterinary pathology in scanning
surveillance for both new and re-emerging animal disease threat detection and characterisation and
for endemic disease monitoring is widely acknowledged, there is a need to further support this aspect
of veterinary pathology39. Elements to consider include research training that translates to problemsolving abilities40, harmonisation of nomenclature and support for rapid knowledge transfer between
pathologists.
Acknowledgements
The author gratefully acknowledges the contributions of many colleagues including particularly K
Willoughby, M Rocchi, F Howie, A Colloff and A Holliman.
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