Large scale transcriptional analysis of MHC class I haplotype diversity in sheep.

Domestic sheep (Ovis aries) have been an important component of livestock agricultural production for thousands of years. Preserving genetic diversity within livestock populations maintains a capacity to respond to changing environments and rapidly evolving pathogens. MHC genetic diversity can influence immune functionality at individual and population levels. Here, we focus on defining functional MHC class I haplotype diversity in a large cohort of Scottish Blackface sheep pre-selected for high levels of MHC class II DRB1 diversity. Using high-throughput amplicon sequencing with three independent sets of barcoded primers we identified 134 MHC class I transcripts within 38 haplotypes. Haplotypes were identified with between two and six MHC class I genes, plus variable numbers of conserved sequences with very low read frequencies. One or two highly transcribed transcripts dominate each haplotype indicative of two highly polymorphic, classical MHC class I genes. Additional clusters of medium, low, and very low expressed transcripts are described, indicative of lower transcribed classical, non-classical and genes whose function remains to be determined.

Contemporary selection on MHC genes in a free-living ruminant population.

Genes within the major histocompatibility complex (MHC) are the most variable identified in vertebrates. Pathogen-mediated selection is believed to be the main force maintaining MHC diversity. However, relatively few studies have demonstrated contemporary selection on MHC genes. Here, we examine associations between MHC variation and several fitness measurements including total fitness and five fitness components, in 3400 wild Soay sheep (Ovis aries) monitored between 1989 and 2012. In terms of total fitness, measured as lifetime breeding success of all individuals born, we found haplotypes named C and D were associated with decreased and increased male total fitness respectively. In terms of fitness components, juvenile survival was associated with haplotype divergence while individual haplotypes (C, D and F) were associated with adult fitness components. Consistent with the increased male total fitness, the rarest haplotype D has increased in frequency throughout the study period more than expected under neutral expectations. Our results demonstrate that contemporary natural selection is acting on MHC class II genes in Soay sheep and that the mode of selection on specific fitness components can be different mode from selection on total fitness.

Associations between MHC class II variation and phenotypic traits in a free-living sheep population.

Pathogen-mediated selection (PMS) is thought to maintain the high level of allelic diversity observed in the major histocompatibility complex (MHC) class II genes. A comprehensive way to demonstrate contemporary selection is to examine associations between MHC variation and individual fitness. As individual fitness is hard to measure, many studies examine associations between MHC variation and phenotypic traits, including direct or indirect measures of adaptive immunity thought to contribute to fitness. Here, we tested associations between MHC class II variation and five phenotypic traits measured in free-living sheep captured in August: weight, strongyle faecal egg count, and plasma IgA, IgE and IgG immunoglobulin titres against the gastrointestinal nematode parasite Teladorsagia circumcincta. We found no association between MHC class II variation and weight or strongyle faecal egg count. We did, however, find associations between MHC class II variation and immunoglobulin levels which varied with isotype, age and sex. Our results suggest associations between MHC and phenotypic traits are more likely to be found for traits more closely associated with pathogen defence than integrative traits such as bodyweight and highlight the association between MHC variation and antibodies in wild populations.

MHC class IIa haplotypes derived by high-throughput SNP screening in an isolated sheep population.

Investigating the current evolutionary processes acting on a highly polymorphic gene region, such as the major histocompatibility complex (MHC), requires extensive population data for both genotypes and phenotypes. The MHC consists of several tightly linked loci with both allelic and gene content variation, making it challenging to genotype. Eight class IIa haplotypes have previously been identified in the Soay sheep (Ovis aries) of St. Kilda using Sanger sequencing and cloning, but no single locus is representative of all haplotypes. Here, we exploit the closed nature of the island population of Soay sheep and its limited haplotypic variation to identify a panel of SNPs that enable imputation of MHC haplotypes. We compared MHC class IIa haplotypes determined by Sanger sequence-based genotyping of 135 individuals to their SNP profiles generated using the Ovine Infinium HD BeadChip. A panel of 11 SNPs could reliably determine MHC diplotypes, and two additional SNPs within the DQA1 gene enabled detection of a recombinant haplotype affecting only the SNPs downstream of the expressed genes. The panel of 13 SNPs was genotyped in 5951 Soay sheep, of which 5349 passed quality control. Using the Soay sheep pedigree, we were able to trace the origin and inheritance of the recombinant SNP haplotype. This SNP-based method has enabled the rapid generation of locus-specific MHC genotypes for large numbers of Soay sheep. This volume of high-quality genotypes in a well-characterized population of free-living sheep will be valuable for investigating the mechanisms maintaining diversity at the MHC.

Intramammary Immunisation Provides Short Term Protection Against Mannheimia haemolytica Mastitis in Sheep.

Mastitis affects both dairy and meat/wool sheep industries with losses due to reductions in milk quality and quantity, increased treatment costs and restricted lamb growth. Effective vaccines would be important tools for mastitis control. However, the development of vaccines against mastitis has proved challenging due to the failure to target protective immunity to the mammary gland. In order to target responses to the mammary gland, this study tested whether local administration directly into the gland through the teat canal or in the udder skin confers protection against an intramammary infection. In this study, we tested a vaccine that confers protection against respiratory disease caused by Mannheimia haemolytica to determine if it also protects against intramammary infection by the same organism. No evidence of protection was observed in animals that received a subcutaneous immunisation in the udder skin, however, intramammary immunisation provided almost complete protection against an experimental challenge administered 7 days post immunisation but not if the challenge was delivered 14 days post immunisation. To investigate further the nature of this variation in response, the somatic cell count and concentration of cytokines Interleukin-1ß, Interleukin-10 and Interleukin-17A was determined in milk over the course of each study. Intramammary immunisation induced an inflammatory response within the mammary gland, characterised by increases in SCC and in the production of cytokines IL-1ß, IL-10, and IL-17A. This response was similar to that observed in un-vaccinated control animals post challenge. The SCC and cytokine levels had returned to levels comparable with un-vaccinated controls prior to challenge at both 7 and 14 days post immunisation. The transient nature of the protective effect is consistent with the priming of an innate antibacterial response within the mammary gland which provides protection against challenge at 7 days but is diminished by 14 days post-vaccination. Further studies are planned to determine the nature of the innate immune mechanisms associated with the protective effect described here to determine whether it may be exploited to improve ruminant udder health.

Novel Presentation of DMV-Associated Encephalitis in a Long-Finned Pilot Whale (Globicephala melas).

Cetacean morbillivirus (CeMV) is an important global cause of morbidity and mortality in cetacean populations, with four pathological presentations including non-suppurative encephalitis. We describe an unusual case of dolphin morbillivirus (DMV)-associated non-suppurative encephalitis in a long-finned pilot whale (Globicephala melas), in which the lesions were orientated on the periventricular white matter and comprised prominent multifocal syncytia formation in the absence of systemic lesions. DMV RNA was detected in brain tissue by qRT-PCR and immunohistochemistry for morbillivirus antigen yielded intense labelling of syncytia in periventricular sites, with sparse involvement of the deeper neuroparenchyma. The pattern of lesions raises the possibility of viral dissemination through the cerebrospinal fluid, as described for canine distemper virus, suggesting that similar pathogenic mechanisms may be implicated in lesion development. Further investigation is required to establish the pathogenesis of CeMV encephalitis and the behaviour of the virus within the central nervous system of cetaceans.

Novel Dermatitis and Relative Viral Nucleic Acid Tissue Loads in a Fin Whale (Balaenoptera physalus) with Systemic Cetacean Morbillivirus Infection.

Cetacean morbilliviruses (CeMVs) are significant causes of mortality in many cetacean species in epizootics and smaller outbreaks. Despite the prominence of skin lesions in seals and terrestrial animals, including humans, affected by other morbilliviruses, they have not been reported in CeMV-infected cetaceans. Here we report CeMV-associated skin lesions in a fin whale (Balaenoptera physalus) with subacute, systemic CeMV infection that live-stranded in Scotland, UK. Grossly, the skin was sloughing in large sheets, presumed due to autolysis, but histological examination showed syncytia, below the dermoepidermal junction, that were strongly immunopositive for morbillivirus antigen, as were syncytia in other organs. By polymerase chain reaction (PCR), the relative load of CeMV-specific RNA was largest in the liver and urinary bladder, even in formalin-fixed, paraffin-wax embedded samples. Levels were low in skin and only detectable in frozen samples. Genetic comparison of the CeMV revealed close alignment with isolates from fin whales from the North Atlantic Ocean and Mediterranean Sea, but that it was distinct from the porpoise CeMV clade. These findings show skin samples can be used to diagnose CeMV infection in cetaceans, highlighting the potential of ante-mortem sampling for monitoring disease in current populations and assessment of changes in host and pathogen genetics.

Antimicrobial resistance in ovine bacteria: A sheep in wolf's clothing?

BACKGROUND: To monitor the prevalence of antimicrobial resistance (AMR), methods for interpretation of susceptibility phenotypes of bacteria are needed. Reference limits to declare resistance are generally based on or dominated by data from human bacterial isolates and may not reflect clinical relevance or wild type (WT) populations in livestock or other hosts. METHODS: We compared the observed prevalence of AMR using standard and bespoke interpretations based on clinical breakpoints or epidemiological cut-offs (ECOFF) using gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) bacteria from sheep as exemplars. Isolates were obtained from a cross-sectional study in three lowland sheep flocks in Scotland, and from a longitudinal study in one flock in Norway. S. aureus (n = 101) was predominantly isolated from milk or mammary glands whilst E. coli (n = 103) was mostly isolated from faecal samples. Disc diffusion testing was used to determine inhibition zone diameters, which were interpreted using either clinical breakpoints or ECOFF, which distinguish the bacterial wild type population from bacteria with acquired or mutational resistance to the compound of interest (non-wild type). Standard ECOFF values were considered as well as sheep-specific values calculated from the data using Normalized Resistance Interpretation (NRI) methodology. RESULTS: The prevalence of AMR as measured based on clinical breakpoints was low, e.g. 4.0% for penicillin resistance in S. aureus. Estimation of AMR prevalence based on standard ECOFFs was hampered by lack of relevant reference values. In addition, standard ECOFFS, which are predominantly based on human data, bisected the normal distribution of inhibition zone diameters for several compounds in our analysis of sheep isolates. This contravenes recommendations for ECOFF setting based on NRI methodology and may lead to high apparent AMR prevalence. Using bespoke ECOFF values based on NRI, S. aureus showed non-wild type for less than 4% of isolates across 13 compounds, and ca. 13% non-wild type for amoxicillin and ampicillin, while E. coli showed non-wild type for less than 3% of isolates across 12 compounds, and ca. 13% non-wild type for tetracyclines and sulfamethoxazole-trimethoprim. CONCLUSION: The apparent prevalence of AMR in bacteria isolated from sheep is highly dependent on interpretation criteria. The sheep industry may want to establish bespoke cut-off values for AMR monitoring to avoid the use of cut-offs developed for other host species. The latter could lead to high apparent prevalence of resistance, including to critically important antimicrobial classes such as 4th generation cephalosporins and carbapenems, suggesting an AMR problem that may not actually exist.

A novel technique for retrospective genetic analysis of the response to vaccination or infection using cell-free DNA from archived sheep serum and plasma.

Genetic variation is associated with differences in disease resistance and susceptibility among individuals within a population. To date, molecular genetic analyses of host responses have relied on extraction of genomic DNA from whole blood or tissue samples. However, such samples are not routinely collected during large-scale field studies. We demonstrate that cell-free genomic DNA (cfDNA) may be extracted and amplified from archived plasma samples, allowing retrospective analysis of host genetic diversity. This technique was also applicable to archived serum samples up to 35 years old and to different ruminant species. As proof of concept, we used this cfDNA approach to genotype the major histocompatibility complex (MHC) class II DRB1 locus of 224 Merino sheep which had participated in field trials of a commercial Haemonchus contortus vaccine, Barbervax®, in Australia. This identified a total of 51 different DRB1 alleles and their relative frequencies. This is the first study to examine host MHC diversity using DNA extracted from archived plasma samples, an approach that may be applied to retrospective analyses of genetic diversity and responses to vaccination or infection across different species and populations.

Characterisation of major histocompatibility complex class IIa haplotypes in an island sheep population.

The ovine MHC class IIa is known to consist of six to eight loci located in close proximity on chromosome 20, forming haplotypes that are typically inherited without recombination. Here, we characterise the class IIa haplotypes within the Soay sheep (Ovis aries) on St. Kilda to assess the diversity present within this unmanaged island population. We used a stepwise sequence-based genotyping strategy to identify alleles at seven polymorphic MHC class IIa loci in a sample of 118 Soay sheep from four cohorts spanning 15 years of the long-term study on St. Kilda. DRB1, the most polymorphic MHC class II locus, was characterised first in all 118 sheep and identified six alleles. Using DRB1 homozygous animals, the DQA (DQA1, DQA2 and DQA2-like) and DQB (DQB1, DQB2 and DQB2-like) loci were sequenced, revealing eight haplotypes. Both DQ1/DQ2 and DQ2/DQ2-like haplotype configurations were identified and a single haplotype carrying three DQB alleles. A test sample of 94 further individuals typed at the DRB1 and DQA loci found no exceptions to the eight identified haplotypes and a haplotype homozygosity of 21.3%. We found evidence of historic positive selection at DRB1, DQA and DQB. The limited variation at MHC class IIa loci in Soay sheep enabled haplotype characterisation but showed that no single locus could capture the full extent of the expressed variation in the region.

Allelic nomenclature for the duplicated MHC class II DQ genes in sheep.

The principal MHC class II molecules involved in the presentation of peptides to the antigen specific receptors on CD4+ T cells genes in sheep are derived from DR and DQ genes. Allelic nomenclature systems for the DRB1 and its partner DRA loci are available for Ovid's; however, no official nomenclature is available for the DQ genes which creates ambiguity within the research community. Ovine MHC haplotypes include at least two pairs of DQA and DQB genes, termed DQA1, DQB1 and DQA2, DQB2 and both sets are polymorphic and both seem to be functional. In a number of haplotypes, the DQA1 locus appears to be absent (DQA1-null) and is replaced by a second locus termed DQA2-like. Here, we identify families of alleles based on sequence similarity and phylogenetic clustering which correspond to each of the DQA and DQB genes identified in previous genomic and transcript analyses of homozygous animals. Using such criteria to cluster sequences, we have named 82 full-length and partial cDNA transcripts derived from domestic sheep (Ovis aries) which correspond to alleles at the Ovar-DQA1, DQA2, DQA2-like, DQB1, DQB2 and DQB2-like genes and provide associated sequence resources available to the research community through the IPD-MHC Database. This sets the basis for naming and annotation of DQ genes within the ovine MHC and may be used as a template for DQ genes in other ruminant species which will ultimately support research in livestock infectious disease.

An official nomenclature for the major histocompatibility complex allele sequences from the domestic goat (Capra hircus).

Official allelic nomenclature and corresponding databases of validated major histocompatibility complex (MHC) alleles from most of the major species of farmed livestock are now represented on the IPD-MHC Database. The major exception is the domestic goat (Capra hircus) which can lead to confusion in the research community. Here, we propose to start the process of developing such a resource which will support the research community's interests in livestock population genetics, infectious disease research, vaccine development and comparative studies. In this manuscript, we assign the official nomenclature for the major transcribed and highly polymorphic MHC class II DRB1 locus. Additional class II loci including DRA and DQ and MHC class I loci will be added in the future.

Comparative MHC nomenclature: report from the ISAG/IUIS-VIC committee 2018.

Significant progress has been made over the last decade in defining major histocompatibility complex (MHC) diversity at the nucleotide, allele, haplotype, diplotype, and population levels in many non-human species. Much of this progress has been driven by the increased availability and reduced costs associated with nucleotide sequencing technologies. This report provides an update on the activities of the comparative MHC nomenclature committee which is a standing committee of both the International Society for Animal Genetics (ISAG) and the International Union of Immunological Societies (IUIS) where it operates under the umbrella of the Veterinary Immunology Committee (VIC). A previous report from this committee in 2006 defined the role of the committee in providing guidance in the development of a standardized nomenclature for genes and alleles at MHC loci in non-human species. It described the establishment of the Immuno Polymorphism Database, IPD-MHC, which continues to provide public access to high quality MHC sequence data across a range of species. In this report, guidelines for the continued development of a universal MHC nomenclature framework are described, summarizing the continued development of each species section within the IPD-MHC project.

IPD-MHC: nomenclature requirements for the non-human major histocompatibility complex in the next-generation sequencing era.

The IPD-MHC Database is the official repository for non-human MHC sequences, overseen and supported by the Comparative MHC Nomenclature Committee, providing access to curated MHC data and associated analysis tools. To address the increasing amount and complexity of data being submitted, an entirely upgraded version of the IPD-MHC Database was recently released to maintain IPD-MHC as the central platform for the comparison of curated MHC data. As a consequence, a new level of nomenclature standardisation is required between the different species to enable data submission and to allow the unambiguous inter- and intra-species comparison of alleles. However, any changes must retain the flexibility demanded by the unique biology of different taxonomic groups. Here, we describe the rationale for a standardised nomenclature system and summarise the changes that have been driven by the requirements of implementing the IPD-MHC database. This modified nomenclature system is essential to maintain the current functionality of IPD-MHC and provide a scalable future-proof database organisation to fully exploit the bioinformatic tools used for analysis.

Structural and functional diversity arising from intra- and inter-haplotype combinations of duplicated DQA and B loci within the ovine MHC.

In sheep, the A and B loci encoding the α and ß chains of the classical class II MHC molecules are DRA and DRB and DQA and DQB. Previous analyses described the duplication of the DQA and DQB genes. The majority of haplotypes include DQA1 and DQA2 loci, however, in a number of haplotypes, DQA1 appears absent and these haplotypes have been described as DQA1 null. In these haplotypes, the DQA2 locus is found in combination with a second locus which appeared more closely related to DQA2 than DQA1, hence the description of this locus as DQA2-like. Here we combine our previous analysis of the DQA transcripts with an analysis of the associated DQB transcripts in ten haplotypes from MHC homozygous animals. This allows the potential for surface expression of different haplotype combinations of DQA and B genes and the functional significance of DQA2-like and its predicted DQB partner to be determined. Atypical DQB transcripts (DQB2-like) were identified in haplotypes classified as DQA1-null and conserved DQB2-like orthologues were identified in other Bovidae indicating trans-species conservation of the allelic lineage. Functional combinations detected by co-transfection of DQ1, DQ2 and DQ2-like genes demonstrates the potential for a wide range of DQ molecules derived from both intra- and inter-haplotype as well as inter-locus combinations. We provide evidence that DQA2-like and B2-like genes form an evolutionary conserved pair which generates structurally distinct class II molecules that are likely to present a distinct range of peptides to CD4+ T cells.

An ancient interlocus recombination increases class II MHC DQA diversity in sheep and other Bovidae.

Animals with fully characterised major histocompatibility complex (MHC) regions are often used to explore the molecular interactions that control the induction of adaptive immunity. The ovine MHC includes two DQA loci, termed DQA1 and DQA2. However, in a minority of haplotypes the DQA1 locus appears absent (DQA1 null) and is replaced by a second locus termed, DQA2-like. This raises a number of questions regarding the origins and function of the DQA2-like sequences. To address this, we have analysed DQA diversity associated with 10 MHC haplotypes, including two classified as DQA1 null. Pair-wise comparison between full-length DQA transcripts from each haplotype identified unique diversity throughout the DQA2-like sequences. Conserved orthologues of the DQA2-like sequences were identified in cattle and goat, and phylogenetic analysis clustered exons 1 and 2 with DQA2 whereas the remainder of the sequence clustered with DQA1. The DQA2-like allelic lineage appears functional and to have arisen from an ancient interlocus recombination between DQA1 and DQA2 loci which predates Bovidae speciation.

Non-human lnc-DC orthologs encode Wdnm1-like protein.

In a recent publication in Science, Wang et al. found a long noncoding RNA (lncRNA) expressed in human dendritic cells (DC), which they designated lnc-DC. Based on lentivirus-mediated RNA interference (RNAi) experiments in human and murine systems, they concluded that lnc-DC is important in differentiation of monocytes into DC. However, Wang et al. did not mention that their so-called "mouse lnc-DC ortholog" gene was already designated " Wdnm1-like" and is known to encode a small secreted protein.  We found that incapacitation of the Wdnm1-like open reading frame (ORF) is very rare among mammals, with all investigated primates except for hominids having an intact ORF. The null-hypothesis by Wang et al. therefore should have been that the human lnc-DC transcript might only represent a non-functional relatively young evolutionary remnant of a protein coding locus.  Whether this null-hypothesis can be rejected by the experimental data presented by Wang et al. depends in part on the possible off-target (immunogenic or otherwise) effects of their RNAi procedures, which were not exhaustive in regard to the number of analyzed RNAi sequences and control sequences.  If, however, the conclusions by Wang et al. on their human model are correct, and they may be, current knowledge regarding the Wdnm1-like locus suggests an intriguing combination of different functions mediated by transcript and protein in the maturation of several cell types at some point in evolution. We feel that the article by Wang et al. tends to be misleading without the discussion presented here.

Comparison of bacteriological culture and PCR for detection of bacteria in ovine milk--sheep are not small cows.

Mastitis, inflammation of the mammary gland, is an important cause of disease, mortality, and production losses in dairy and meat sheep. Mastitis is commonly caused by intramammary infection with bacteria, which can be detected by bacterial culture or PCR. PathoProof (Thermo Fisher Scientific Ltd., Vantaa, Finland) is a commercially available real-time PCR system for the detection of bovine mastitis pathogens. Sheep differ from cattle in the bacterial species or bacterial strains that cause mastitis, as well as in the composition of their milk. The aim of this study was to evaluate whether the PathoProof system was suitable for detection of mastitis pathogens in sheep milk. Milk samples were collected aseptically from 219 udder halves of 113 clinically healthy ewes in a single flock. Aliquots were used for bacteriological culture and real-time PCR-based detection of bacteria. For species identified by culture, the diagnosis was confirmed by species-specific conventional PCR or by sequencing of a housekeeping gene. The majority of samples were negative by culture (74.4% of 219 samples) and real-time PCR (82.3% of 192 samples). Agreement was observed for 138 of 192 samples. Thirty-four samples were positive by culture only, mostly due to presence of species that are not covered by primers in the PCR system (e.g., Mannheimia spp.). Two samples were positive for Streptococcus uberis by culture but not by PCR directly from the milk samples. This was not due to inability of the PCR primers to amplify ovine Streptococcus uberis, as diluted DNA extracts from the same samples and DNA extracts from the bacterial isolates were positive by real-time PCR. For samples containing Staphylococcus spp., 11 samples were positive by culture and PCR, 9 by culture only, and 20 by PCR only. Samples that were negative by either method had lower bacterial load than samples that were positive for both methods, whereas no clear relation with species identity was observed. This study provides proof of principle that real-time PCR can be used for detection of mastitis pathogens in ovine milk. Routine use in sheep may require inclusion of primer sets for sheep-specific mastitis pathogens.

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.

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.