Three cases of alloimmune mediated pancytopenia in calves resembling bovine neonatal pancytopenia

Background Between 2007 and 2011 several thousands of calves died from bovine neonatal pancytopenia (BNP), a bleeding syndrome triggered by vaccine induced alloantibodies from the dams. Following withdrawal of the involved bovine viral diarrhoea virus (BVDv) vaccine, the incidence of this condition rapidly decreased, with no reported cases in the last 5 years. Here, we report a recent immune-mediated pancytopenia in three calves from two different suckler herds, clinically indistinguishable from BNP. Case presentation Three Belgian Blue suckler calves from two different farms, aged around two weeks, showed multiple bleedings disseminated on the skin and petechiae and ecchymoses on the mucosae. Blood examination confirmed anaemia, leukopenia and thrombocytopenia. BVDv infection was excluded. Despite blood transfusion and cortisone therapy, all three animals died. Necropsy and histology confirmed bone marrow depletion. Binding of IgG from the dams on leukocytes of the calves was demonstrated by flow cytometry. Two calves, originating from the same farm, received colostrum from the same dam. None of the calves were given colostrum replacers or colostrum supplements. No link with the BNP causing BVDv vaccine could be evidenced. However, dams had been vaccinated against bovine herpesvirus 1, parainfluenza-3 virus, bovine respiratory syncytial virus and bluetongue virus serotype 8. Conclusions Alloimmune mediated pancytopenia was evidenced in three animals, clinically and pathologically indistinguishable from BNP. Whether this disease is again vaccine mediated remains to be determined.

Detection of Bovine Herpesvirus 1 from Semen by Real-time PCR to Prevent the Spread of Infectious Bovine Rhinotracheitis Infection

Infectious Bovine Rhinotracheitis (IBR) can be transmitted by livestock seeds and semen, through the process of artificial insemination. Therefore, it is necessary to detect the presence of the Bovine Herpesvirus 1 (BHV1) in semen through artificial insemination and breeding centers of Indonesia. The current study aimed to detect the presence of the virus in semen as a source of IBR disease transmission in Indonesia. A total of 27 semen samples from artificial insemination and breeding centers (Sembawa, Lembang, Ungaran, and Sleman) in Indonesia have been examined and identified using the real-time PCR (qPCR) technique. The result showed that all samples were negative to BHV1. This indicated that semen from Ungaran, Sembawa, Sleman, and Lembang was safe to be used as a semen source for artificial insemination.

Longitudinal Prevalence of Antibodies to Endemic Pathogens in Bulk Tank Milk Samples From Dairy Herds Engaged or Not in Contract Heifer Rearing

Since the abolition of EU milk production quotas in 2015, Europe's dairy industries have undergone a period of rapid expansion with possible resultant increased inter-herd transmission of endemic pathogens. The aims of this study were (1) to establish the post-2015 prevalence of antibodies to selected endemic infectious diseases and (2) to determine if prevalences differed between herds where heifers were reared at home and those where heifers were sent out for contract-rearing. Three bulk tank milk (BTM) samples were collected annually between May and August of 2018–20 inclusively from 120 Irish dairy herds. Additionally, herd vaccination status was collected by questionnaire. Milk samples were tested using commercially available ELISAs for eight pathogens: bovine viral diarrhea virus (BVDV), bovine herpesvirus 1 (BoHv-1), bovine respiratory syncytial virus (BRSV), Mycoplasma bovis, Mycobacterium avium subspecies paratuberculosis (MAP), Salmonella Dublin (S. Dublin), Leptospira Hardjo (L. Hardjo), and Neospora caninum (N. caninum). The true prevalence of each pathogen was calculated using a Rogan-Gladen estimator. The true prevalences (95% CI) of BTM antibodies in unvaccinated herds across the 3 years were as follows (i) BVDV: 57, 86, and 73% (95% CI: 40.7–65.9, 74–94, and 58–85) (n = 56, 56, and 48), (ii) BoHv-1: 47, 49, and 19% (95% CI: 26.3–69.7, 25–75, and 1–56) (n = 21, 20, and 11), (iii) L. Hardjo: 34, 59, and 73% (95% CI: 12.5–63, 33–82, and 33–99) (n = 15, 21, and 10), (iv) S. Dublin 32, 57, and 11% (95% CI: 12.21–68.1, 30.2–90.1, and 0) (n = 19, 22, and 13), (v) BRSV: 100% (95% CI: 99.5–100, 100, and 100) (n = 120, 109, and 91), (vi) MAP: 0% (95% CI: 0, 0, and 0) (n = 120, 109, and 91) (vii) N. caninum 0% (95% CI: 0, 0, and 0) (n = 120, 109, and 91) and (viii) M. bovis (ELISA) 53, 0.42, and 30% (95% CI: 3.95–6.84, 0, and 21–41) (n = 120, 109, and 91). M. bovis was detected by PCR in 0, 1, and 0% of herds in 2018, 2019, and 2020, respectively. This study showed that expanding Irish dairy herds are endemically infected with several of the studied pathogens. No differences in herd prevalence of infectious agents were observed between farms with different heifer rearing strategies (contract-rearing vs. traditional rearing).

Risk Factors for Introduction of Bovine Herpesvirus 1 (BoHV-1) Into Cattle Herds: A Systematic European Literature Review

Given that bovine herpesvirus 1 (BoHV-1) -the causative agent of Infectious Bovine Rhinotracheitis (IBR)- is still endemic in most European countries, BoHV-1 free herds are subject to a considerable risk of (re)introduction of the virus. The aim of this literature review was to describe published, quantified risk factors that are relevant for the introduction of BoHV-1. The risk factors described in this study can be used as input for modeling eradication scenarios and for communication on biosecurity measures to stakeholders. A literature search was conducted in November 2020 in two major online search databases, PubMed and Web of Science. The search criteria “risk factor” combined with different synonyms for BoHV-1 were explored, which resulted in 564 hits. Only studies performed in Europe, written in Dutch, English, French, German or Spanish with an English summary and that quantified risk factors for introduction of BoHV-1 into cattle herds were included. Studies had to quantify the risk factors with crude odds ratios (OR), an estimate of the chance of a particular event occurring in an exposed group to a non-exposed group. After checking for duplicates and excluding articles that did not meet the inclusion criteria, 12 publications remained for this review. Risk factors were classified into seven groups, i.e., herd characteristics, management, animal characteristics, purchase, direct animal contact, neighborhood and indirect transmission routes. Most relevant factors for introduction of BoHV-1 into cattle herds include herd size, purchase of cattle, cattle density, age of cattle, distance to neighboring cattle herds and professional visitors. Together with other direct and indirect animal contacts, these factors are important when elimination of BoHV-1 is considered. A closed farming system and protective clothing for professional visitors can eliminate the major routes of introduction of BoHV-1 in cattle herds. To the best of our knowledge, this is the first systematic review solely focussing on measures that can be taken to control introduction of BoHV-1 into cattle herds. Besides testing, focus on managing these (biosecurity) factors will decrease the risk of introducing the virus.

Assessment of bovine respiratory disease progression in calves challenged with bovine herpesvirus 1 and Mannheimia haemolytica using point-of-care and laboratory-based blood leukocyte differential assays

Blood leukocyte differentials can be useful for understanding changes associated with bovine respiratory disease (BRD) progression. By improving turnaround time, point-of-care leukocyte differential assays (PCLD) may provide logistical advantages to laboratory-based assays. Our objective was to assess BRD progression in steers challenged with bovine herpesvirus 1 and Mannheimia haemolytica using point-of-care and laboratory-based blood leukocyte differentials. Thirty Holstein steers (average body weight of 211 kg + 2.4 kg) were inoculated intranasally on day 0 with bovine herpesvirus 1 and intrabronchially on day 6 with Mannheimia haemolytica. Blood leukocytes differentials were measured using both assays from study day 0 to 13. Linear mixed models were fitted to evaluate the associations between: 1) the type of assay (laboratory-based or PCLD) with respect to leukocyte, lymphocyte, and neutrophil concentrations, 2) study day with cell concentrations, and 3) cell concentrations with lung consolidation measured at necropsy. Point-of-care leukocyte, lymphocyte, and neutrophil concentrations were significantly associated (P < 0.05) with the respective cell concentrations obtained from the laboratory-based leukocyte differential. Cell concentrations reported by both assays differed significantly (P < 0.05) over time, indicating shifts from healthy to viral and bacterial disease states. Lymphocyte concentrations, lymphocyte / neutrophil ratios obtained from both assays, and band neutrophil concentrations from the laboratory-based assay were significantly associated (P < 0.05) with lung consolidation, enhancing assessments of disease severity. The PCLD may be a useful alternative to assess BRD progression when laboratory-based leukocyte differentials are impractical.

VP8, the Major Tegument Protein of Bovine Herpesvirus-1, Is Partially Packaged during Early Tegument Formation in a VP22-Dependent Manner

Bovine herpesvirus-1 (BoHV-1) is a major cause of rhinotracheitis and vulvovaginitis in cattle. VP8, the major tegument protein of BoHV-1, is essential for viral replication in the host. VP8 is phosphorylated by the viral kinase US3, mediating its translocation to the cytoplasm. VP8 remains nuclear when not phosphorylated. Interestingly, VP8 has a significant presence in mature BoHV-1YmVP8, in which the VP8 phosphorylation sites are mutated. This suggests that VP8 might be packaged during primary envelopment of BoHV-1. This was investigated by mass spectrometry and Western blotting, which showed VP8, as well as VP22, to be constituents of the primary enveloped virions. VP8 and VP22 were shown to interact via co-immunoprecipitation experiments, in both BoHV-1-infected and VP8-transfected cells. VP8 and VP22 also co-localised with one another and with nuclear lamin-associated protein 2 in BoHV-1-infected cells, suggesting an interaction between VP8 and VP22 in the perinuclear region. In cells infected with VP22-deleted BoHV-1 (BoHV-1ΔUL49), VP8 was absent from the primary enveloped virions, implying that VP22 might be critical for the early packaging of VP8. In conclusion, a novel VP22-dependent mechanism for packaging of VP8 was identified, which may be responsible for a significant amount of VP8 in the viral particle.

Oncolytic Bovine Herpesvirus 1 Inhibits Human Lung Adenocarcinoma A549 Cell Proliferation and Tumor Growth by Inducing DNA Damage

Bovine herpesvirus 1 (BoHV-1) is a promising oncolytic virus with broad antitumor spectrum; however, its oncolytic effects on human lung adenocarcinoma in vivo have not been reported. In this study, we report that BoHV-1 can be used as an oncolytic virus for human lung adenocarcinoma, and elucidate the underlying mechanism of how BoHV-1 suppresses tumor cell proliferation and growth. First, we examined the oncolytic activities of BoHV-1 in human lung adenocarcinoma A549 cells. BoHV-1 infection reduced the protein levels of histone deacetylases (HDACs), including HDAC1-4 that are promising anti-tumor drug targets. Furthermore, the HDAC inhibitor Trichostatin A (TSA) promoted BoHV-1 infection and exacerbated DNA damage and cytopathology, suggesting a synergy between BoHV-1 and TSA. In the A549 tumor xenograft mouse model, we, for the first time, showed that BoHV-1 can infect tumor and suppressed tumor growth with a similar high efficacy as the treatment of TSA, and HDACs have potential effects on the virus replication. Taken together, our study demonstrates that BoHV-1 has oncolytic effects against human lung adenocarcinoma in vivo.

A pioneer transcription factor and type I nuclear hormone receptors synergistically activate the bovine herpesvirus 1 infected cell protein 0 (ICP0) early promoter.

Following bovine herpesvirus 1 (BoHV-1) acute infection of ocular, oral or nasal cavities, sensory neurons within trigeminal ganglia are an important site for latency. Stress, as mimicked by the synthetic corticosteroid dexamethasone, consistently induces reactivation from latency. Expression of two key viral transcriptional regulatory proteins, infected cell protein 0 (bICP0) and bICP4, are regulated by sequences within the immediate early promoter (IEtu1). A separate early promoter also drives bICP0 expression, presumably to ensure sufficient levels of this important transcriptional regulatory protein. Productive infection and bICP0 early promoter activity are cooperatively transactivated by Krüppel like factor 4 (KLF4) and a Type I nuclear hormone receptor (NHR), androgen receptor, glucocorticoid receptor, or progesterone receptor. The bICP0 early promoter contains 3 separate transcriptional enhancers that mediate cooperative transactivation. In contrast to the IEtu1 promoter, the bICP0 early promoter lacks consensus Type I NHR binding sites. Consequently, we hypothesized KLF4 and Sp1 binding sites are essential for Type I NHR and KLF4 to transactivate the bICP0 promoter. Mutating KLF4 and Sp1 binding sites in each enhancer domain significantly reduced transactivation by KLF4 and a Type I NHR. Chromatin immunoprecipitation (ChIP) studies demonstrated occupancy of bICP0 early promoter sequences by KLF4 and Type I NHR is significantly reduced when KLF4 and/or Sp1 binding sites were mutated. These studies suggest cooperative transactivation of the bICP0 E promoter by Type I NHRs and a stress induced pioneer transcription factor (KLF4) promote viral replication and spread in neurons or non-neural cells in reproductive tissue. IMPORTANCE Understanding how stressful stimuli and changes in cellular milieu mediate viral replication and gene expression in the natural host is important for developing therapeutic strategies that impair virus transmission and disease. For example, bovine herpesvirus 1 (BoHV-1) reactivation from latency is consistently induced by the synthetic corticosteroid dexamethasone, which mimics the effects of stress. Furthermore, BoHV-1 infection increases the incidence of abortion in pregnant cows suggesting sex hormones stimulate viral growth in certain tissue. Previous studies revealed Type I nuclear hormone receptors (androgen, glucocorticoid, or progesterone) and the pioneer transcription factor, Krüppel like factor 4 (KLF4), cooperatively transactivate the BoHV-1 infected cell protein 0 (bICP0) early promoter. Transactivation was mediated by Sp1 and/or KLF4 consensus binding sites within the 3 transcriptional enhancers. These studies underscore the complexity by which BoHV-1 exploits Type I NHR fluctuations to enhance viral gene expression, replication, and transmission in the natural host.