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.     

Virus Neutralizing Antibodies Against a Panel of 18 BVDV Isolates in Calves Vaccinated with Rispoval™ RS‐BVD

Seven of nine colostrum‐deprived calves, free from infection with bovine virus diarrhoea virus (BVDV), were vaccinated with Rispoval^? RS‐BVD on two occasions, 21 days apart, while the other two were kept as BVDV infection controls. The virus neutralizing (VN) serum antibodies induced by vaccination were tested for their ability to neutralize 18 European BVDV isolates, including laboratory reference strains and recent field isolates, both cytopathic and non‐cytopathic biotypes as well as genotypes I and II. The strains were isolated in Belgium, France, Germany and the United Kingdom. While there were large variations in the vaccine‐induced VN titres of the individual calves against all the strains, e.g. the titres against Osloss NCP, the European reference strain ranged from 1.7 to 6.7 (1 : log₂), serum from each animal was capable of neutralizing between nine and all 18 of the strains tested. Nevertheless, from the results of this study, it can be concluded that in colostrum‐deprived BVDV seronegative calves, Rispoval^? RS‐BVD can stimulate the production of VN antibodies capable of neutralizing a wide range of antigenically diverse European isolates of BVDV, including genotypes I and II.     

Effect of maternal cells transferred with colostrum on cellular responses to pathogen antigens in neonatal calves

OBJECTIVE: To assess the effect of maternal cells or cellular components on neonatal immune responses to intracellular pathogens in calves. ANIMALS: 15 Holstein calves. PROCEDURES: Calves were fed whole colostrum, frozen colostrum, or cell-free colostrum within 4 hours after birth. Leukocytes were obtained from calves before feeding colostrum and 1, 2, 7, 14, 21, and 28 days after ingestion. Proliferative responses against bovine viral diarrhea virus (BVDV) and mycobacterial purified protein derivatives were evaluated. Dams received a vaccine containing inactivated BVDV, but were not vaccinated against mycobacterial antigens. RESULTS: All calves had essentially no IgG in circulation at birth, but comparable and substantial concentrations by day 1. Calves that received whole colostrum had enhanced responses to BVDV antigen 1 and 2 days after ingestion of colostrum. In contrast, calves that received frozen colostrum or cell-free colostrum did not respond to BVDV. No differences were identified among the 3 groups in response to mycobacterial antigens. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that transfer of live maternal cells from colostrum to neonatal calves enhanced responses to antigens against which the dams had previously responded (BVDV), but not to antigens to which the dams were na?ve (mycobacterial purified protein derivatives). Results suggested that cell-mediated immune transfer to neonates can be enhanced by maternal vaccination.     

Virus neutralising antibodies against 22 bovine viral diarrhoea virus isolates in vaccinated calves.

Seven of nine colostrum deprived calves, free from bovine viral diarrhoea virus (BVDV), were vaccinated with a commercially available vaccine containing two inactivated strains of BVDV, an inactivated strain of bovine herpesvirus-1 and modified-live strains of bovine respiratory syncytial virus and para-influenza-3 virus. The two other calves were kept as controls. The virus neutralising (VN) antibodies induced by vaccination were tested against 22 antigenically diverse BVDV isolates, including reference strains and field isolates, both cytopathic and non-cytopathic, as well as genotypes I and II. The strains were isolated in Belgium, France, Germany, the United Kingdom and the USA. While there were variations in the VN titres of the individual calves against all the strains, serum from the seven animals neutralised 20 or more of the strains tested. From the results, it can be concluded that the vaccine can stimulate the production of VN antibodies capable of neutralising a wide range of European and American isolates of BVDV, including genotypes I and II.     

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

ABSTRACT: 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 β-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.     

Effect of passive immunity on the development of a protective immune response against bovine viral diarrhea virus in calves

OBJECTIVE: To determine whether passively acquired antibodies prevent development of a protective immune response to live virus in calves. ANIMALS: 18 calves. PROCEDURES: Calves were caught immediately after birth and tested free of bovine viral diarrhea virus (BVDV) and serum antibodies against BVDV. Within 48 hours, 12 calves were fed colostrum that contained antibodies against BVDV and 6 calves received BVDV antibody free milk replacer. Three milk replacer fed and 6 colostrum fed calves were exposed to virulent BVDV2-1373 at 2 to 5 weeks of life when passively acquired serum antibody titers were high. After serum antibody titers against BVDV had decayed to undetectable concentrations (at 7 to 9 months of age), the 3 remaining milk replacer fed calves, 6 colostrum fed calves previously exposed to BVDV2-1373, and 6 colostrum fed calves that had not been exposed to the virus were inoculated with BVDV2-1373. RESULTS: Passively acquired antibodies prevented clinical disease in inoculated colostrum fed calves at 2 to 5 weeks of life. Serum antibody titers did not increase in these calves following virus inoculation, and serum antibody titers decayed at the same rate as in noninoculated colostrum fed calves. Inoculated colostrum fed calves were still protected from clinical disease after serum antibody titers had decayed to nondetectable concentrations. Same age colostrum fed calves that had not been previously exposed to the virus were not protected. CONCLUSIONS AND CLINICAL RELEVANCE: A protective immune response was mounted in calves with passive immunity, but was not reflected by serum antibodies titers. This finding has implications for evaluating vaccine efficacy and immune status.     

Quantification,risk factors,and health impact of natural congenital infection with bovine viral diarrhea virus in dairy calves

OBJECTIVES: To estimate risk and identify risk factors for congenital infection with bovine viral diarrhea virus (BVDV) not resulting in persistent infection and examine effect of congenital infection on health of dairy calves. ANIMALS: 466 calves. PROCEDURES: Calves from 2 intensively managed drylot dairies with different vaccination programs and endemic BVDV infection were sampled before ingesting colostrum and tested with their dams for BVDV and BVDV serum-neutralizing antibodies. Records of treatments and death up to 10 months of age were obtained from calf ranch or dairy personnel. Risk factors for congenital infection, including dam parity and BVDV titer, were examined by use of logistic regression analysis. Effect of congenital infection on morbidity and mortality rates was examined by use of survival analysis methods. RESULTS: Fetal infection was identified in 10.1% of calves, of which 0.5% had persistent infection and 9.6% had congenital infection. Although dependent on herd, congenital infection was associated with high BVDV type 2 titers in dams at calving and with multiparous dams. Calves with congenital infection had 2-fold higher risk of a severe illness, compared with calves without congenital infection. CONCLUSIONS AND CLINICAL RELEVANCE: The unexpectedly high proportion of apparently healthy calves found to be congenitally infected provided an estimate of the amount of fetal infection via exposure of dams and thus virus transmission in the herds. Findings indicate that congenital infection with BVDV may have a negative impact on calf health, with subsequent impact on herd health.     

Virus neutralizing antibodies against a panel of 18 BVDV isolates in calves vaccinated with Rispoval RS-BVD

Seven of nine colostrum-deprived calves, free from infection with bovine virus diarrhoea virus (BVDV), were vaccinated with Rispoval RS-BVD on two occasions, 21 days apart, while the other two were kept as BVDV infection controls. The virus neutralizing (VN) serum antibodies induced by vaccination were tested for their ability to neutralize 18 European BVDV isolates, including laboratory reference strains and recent field isolates, both cytopathic and non-cytopathic biotypes as well as genotypes I and II. The strains were isolated in Belgium, France, Germany and the United Kingdom. While there were large variations in the vaccine-induced VN titres of the individual calves against all the strains, e.g. the titres against Osloss NCP, the European reference strain ranged from 1.7 to 6.7 (1:log2), serum from each animal was capable of neutralizing between nine and all 18 of the strains tested. Nevertheless, from the results of this study, it can be concluded that in colostrum-deprived BVDV seronegative calves, Rispoval RS-BVD can stimulate the production of VN antibodies capable of neutralizing a wide range of antigenically diverse European isolates of BVDV, including genotypes I and II.     

Sera from dams of calves with bovine neonatal pancytopenia contain alloimmune antibodies directed against calf leukocytes

Bovine neonatal pancytopenia (BNP) is a bleeding and pancytopenic syndrome in neonatal calves, which recently emerged all over Europe. The present study tested whether antibodies directed against calf leukocytes are present in sera from known BNP dams. Sera from BNP dams (n=11) were combined with leukocytes from 11 calves (5 BNP survivors and 6 controls). After adding a fluorescein conjugated F(ab')(2) fragment of rabbit anti-bovine IgG (H&L) the level of antibody binding was measured by flow cytometry. As control groups both sera from dams from BNP affected (n=48) as from unaffected (n=54) herds were combined with leukocytes from the same calves. With sera from BNP dams, antibody binding could be visualised by immunofluoresence in both peripheral blood as in bone marrow smears. Mean fluoresence intensity values of all leukocyte subpopulations were significantly higher for the BNP dams compared to both control groups (P<0.01). BNP dams showed significantly more antibody binding on multiple leukocyte subpopulations of both BNP survivors and control calves and this from cut off values of MFI 100 onwards (P<0.01). The BNP survivor calves reacted significantly more often with sera from the BNP dams than the control calves (P<0.01). In conclusion the present study supports the hypothesis that BNP is an immune-mediated disease.     

Vertical transmission of bovine leukemia virus and bovine immunodeficiency virus in dairy cattle herds.

Vertical transmission of bovine leukemia virus (BLV) and bovine immunodeficiency virus (BIV) was investigated in five dairy cattle herds in Hokkaido, where 36.1 and 17.0% of cattle were BLV and BIV seropositive, respectively, and 9.9% of dams were co-infected with both BIV and BLV. Twenty six cases of offspring born from dams infected with only BLV (17 cases) or with both BIV and BLV (9 cases) were examined for the presence of BLV and BIV before and after colostrum feeding by polymerase chain reaction (PCR) and syncytium assay. After birth, all calves were separated immediately from their dams. The offspring born from BLV-positive dams were BLV-negative before colostrum feeding, suggesting that no transplacental transmission had occurred. Thereafter, these offspring were fed colostrum or milk from their dams, but still remained BLV-negative. The other offspring born from BLV-positive dams were fed with BLV-negative colostrum, or with pasteurized BLV-positive colostrum. All these calves remained negative for BLV infection, suggesting that in utero transmission of BLV is negligible. In the case of offspring born from dams co-infected with BLV and BIV, calves were BIV-positive before colostrum feeding at 1 day after the birth, indicating in utero transmission of BIV. After colostrum feeding from their dams, newborn calves became BLV-positive. In addition, one calf was BLV-positive even before colostrum feeding. These results suggest that BIV can be transmitted to offspring in utero, and that BLV can be transmitted through colostrum or milk if dams are infected with both BIV and BLV.     

Increased risk of BVDV infection of calves from pregnant dams on communal Alpine pastures in Switzerland

Skin biopsies and blood samples from 117 calves, the offspring of dams that had been pastured on communal Alpine pastures while pregnant, were examined for bovine viral diarrhoea virus (BVDV) antigen. Immunohistochemical evaluation of skin biopsy samples revealed BVDV antigen in nine (7.7%) calves, and ELISA testing of serum samples was positive for BVDV antigen in six (5.1%). Three calves with positive skin biopsy samples and negative serum results were < 11 days old; it was assumed that maternal antibody interfered with the ELISA testing. Serum samples that were collected at a later date from two of the three calves were positive for BVDV antigen. These results were significantly different from those of a previous study in which the prevalence of persistently infected calves in an average Swiss cattle population was 0.64%. It was concluded that the risk of infection with BVDV is high in cattle sharing a communal Alpine pasture.     

Evaluation of protection against virulent bovine viral diarrhea virus type 2 in calves that had maternal antibodies and were vaccinated with a modified-live vaccine

OBJECTIVE: To evaluate the efficacy of an adjuvanted modified-live bovine viral diarrhea virus (BVDV) vaccine against challenge with a virulent type 2 BVDV strain in calves with or without maternal antibodies against the virus. DESIGN: Challenge study. ANIMALS: 23 crossbred dairy calves. PROCEDURES: Calves were fed colostrum containing antibodies against BVDV or colostrum without anti-BVDV antibodies within 6 hours of birth and again 8 to 12 hours after the first feeding. Calves were vaccinated with a commercial modified-live virus combination vaccine or a sham vaccine at approximately 5 weeks of age and challenged with virulent type 2 BVDV 3.5 months after vaccination. Clinical signs of BVDV infection, development of viremia, and variation in WBC counts were recorded for 14 days after challenge exposure. RESULTS: Calves that received colostrum free of anti-BVDV antibodies and were vaccinated with the sham vaccine developed severe disease (4 of the 7 calves died or were euthanatized). Calves that received colostrum free of anti-BVDV antibodies and were vaccinated and calves that received colostrum with anti-BVDV antibodies and were vaccinated developed only mild or no clinical signs of disease. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the modified-live virus vaccine induced a strong protective immune response in young calves, even when plasma concentrations of maternal antibody were high. In addition, all vaccinated calves were protected against viral shedding, whereas control calves vaccinated with the sham vaccine shed virus for an extended period of time.     

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.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0129-0) contains supplementary material, which is available to authorized users.     

Fetal protection following exposure to calves persistently infected with bovine viral diarrhea virus type 2 sixteen months after primary vaccination of the dams

This study demonstrated that the bovine viral diarrhea virus (BVDV; types 1 and 2) fractions of a multivalent vaccine protected pregnant heifers and their fetuses at 149 to 217 days of gestation against exposure to calves persistently infected with BVDV type 2a. Eighty percent (eight of 10) of the control heifers were viremic at least 1 day following challenge, whereas all (20 of 20) BVDV-vaccinated heifers were virus isolation-negative on all postchallenge assessment days. Ninety percent (nine of 10) of the calves born to control heifers but only 5% (one of 20) of calves born to BVDV-vaccinated heifers seroconverted to BVDV type 2 before ingesting colostrum. One calf born to a control heifer was persistently infected. No calves from BVDV-vaccinated heifers were persistently infected.     

Transplacental and oral transmission of wild-type bluetongue virus serotype 8 in cattle after experimental infection

Potential vertical transmission of wild-type bluetongue virus serotype 8 (BTV-8) in cattle was explored in this experiment. We demonstrated transplacental transmission of wild-type BTV-8 in one calf and oral infection with BTV-8 in another calf. Following the experimental BTV-8 infection of seven out of fifteen multi-parous cows eight months in gestation, each newborn calf was tested prior to colostrum intake for transplacental transmission of BTV by RRT-PCR. If transplacental transmission was not established the calves were fed colostrum from infected dams or colostrum from non-infected dams spiked with BTV-8 containing blood. One calf from an infected dam was born RRT-PCR positive and BTV-specific antibody (Abs) negative, BTV was isolated from its blood. It was born with clinical signs resembling bluetongue and lived for two days. Its post-mortem tissue suspensions were RRT-PCR positive. Of the seven calves fed colostrum from infected dams, none became infected. Of the six calves fed colostrum from non-infected dams spiked with infected blood, one calf became PCR-positive at day 8 post-partum (dpp), seroconverted 27 days later, and remained RRT-PCR and Abs positive for the duration of the experiment (i.e., 70 dpp). This work demonstrates that transplacental transmission in late gestation and oral infection of the neonate with wild-type BTV-8 is possible in cattle under experimental conditions.     

Effect of maternal antibodies on induction and persistence of vaccine-induced immune responses against bovine viral diarrhea virus type II in young calves

OBJECTIVE: To determine the effect of maternally derived antibodies on induction of protective immune responses against bovine viral diarrhea virus (BVDV) type II in young calves vaccinated with a modified-live bovine viral diarrhea virus (BVDV) type I vaccine. DESIGN: Blinded controlled challenge study. ANIMALS: 24 neonatal Holstein and Holstein-cross calves that were deprived of maternal colostrum and fed pooled colostrum that contained a high concentration of (n = 6) or no (18) antibodies to BVDV. PROCEDURE: At 10 to 14 days of age, 6 seropositive and 6 seronegative calves were given a combination vaccine containing modified-live BVDV type I. All calves were kept in isolation for 4.5 months. Six calves of the remaining 12 untreated calves were vaccinated with the same combination vaccine at approximately 4 months of age. Three weeks later, all calves were challenged intranasally with a virulent BVDV type II. RESULTS: Seronegative unvaccinated calves and seropositive calves that were vaccinated at 2 weeks of age developed severe disease, and 4 calves in each of these groups required euthanasia. Seronegative calves that were vaccinated at 2 weeks or 4 months of age developed only mild or no clinical signs of disease. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that a single dose of a modified-live BVDV type-I vaccine given at 10 to 14 days of age can protect susceptible young calves from virulent BVDV type II infection for at least 4 months, but high concentrations of BVDV-specific maternally derived antibodies can block the induction of the response.     

Bovine coronavirus as the causative agent of winter dysentery: serological evidence.

Sera from 9 dairy herds with epizootic enteritis (winter dysentery) were examined for antibodies to bovine coronavirus (BCV) and bovine virus diarrhoea virus (BVDV). Cows in 8 of the 9 herds seroconverted to BCV alone, while the animals in the ninth herd, which showed severe symptoms of the disease, seroconverted both to BCV and BVDV. The BCV antibodies, which were present in high titres 1 year postinfection, were transferred to the offspring via the colostrum and were then detectable in sera of calves until these were approximately 5 months old. A serological survey of 549 Swedish heifers showed that 61% of the animals were reactors to BCV. The prevalence of seroreactors to BCV was equally distributed over Sweden but was commonly either high or low in herds. In conclusion, BCV is commonly detected in animals suffering from winter dysentery. A co-infection with BVDV appears to aggravate the disease.     

Prevalence of bovine viral diarrhea virus (BVDV) in persistently infected cattle and BVDV subtypes in affected cattle in beef herds in south central United States

The prevalence of bovine viral diarrhea virus (BVDV) in persistently infected (PI) cattle in beef breeding herds was determined using 30 herds with 4530 calves. The samples were collected by ear notches and tested for BVDV antigens using immunohistochemistry (IHC) and antigen capture enzyme-linked immunosorbent assay (ACE). Animals with initial positives on both IHC and ACE were sampled again using both tests and serums were collected for viral propagation and sequencing of a viral genomic region, 5′-untranslated region (5′-UTR) for viral subtyping. Samples were also collected from the dams of PI calves. There were 25 PI calves from 4530 samples (0.55%) and these PI calves were from 5 of the 30 herds (16.7%). Two herds had multiple PI calves and 3 herds had only 1 PI calf. Only 1 of the 25 dams with a PI calf was also PI (4.0%). The subtype of all the PI isolates was BVDV1b. Histories of the ranches indicated 23 out of 30 had herd additions of untested breeding females. Twenty-four of the 30 herds had adult cowherd vaccinations against BVDV, primarily using killed BVDV vaccines at pregnancy examination.     

Effect on hematopoietic tissue of experimental infection of calves with noncytopathic type 2 bovine viral diarrhea virus

To investigate the hematologic abnormalities observed with noncytopathic type 2 bovine viral diarrhea virus (ncpBVDV-2), calves 6 to 8 mo old were inoculated with an isolate of either high virulence (HV24515) or low virulence (LV11Q); control animals received the same volume of uninfected cell-culture supernatant. Peripheral blood neutrophil, lymphocyte, and platelet counts decreased in all the virus-inoculated calves but were significantly lower and remained decreased longer in the calves given HV24515. For each isolate, a decrease in the number of mature myeloid cells in the bone marrow coincided with the development of neutropenia, but the depletion persisted significantly longer (4 to 6 d) in the calves given HV24515. In the bone marrow of calves given LV11Q, the number of proliferating myeloid cells increased in proportion to the decrease in the number of mature myeloid cells. In the calves inoculated with HV24515, BVDV antigen was observed in bone marrow cells when the peripheral blood counts were lowest. Megakaryocytes were the predominant cell type exhibiting positive BVDV staining; myeloid cells rarely stained positively. Viral antigen was not observed in the bone marrow of calves given LV11Q. These experiments demonstrated that ncpBVDV-2 isolates of both high and low virulence caused decreased leukocyte and platelet counts, but only the high-virulence HV24515 isolate caused a delay in the production of myeloid proliferating cells. The delay may contribute to the ability of certain ncpBVDV-2 isolates to induce severe disease.