Sandro Cavirani

Bovine herpesvirus 4 is tropic for bovine endometrial cells and modulates endocrine function

Bovine postpartum uterine disease, metritis, affects about 40% of animals and is widely considered to have a bacterial aetiology. Although the gamma-herpesvirus bovine herpesvirus 4 (BoHV-4) has been isolated from several outbreaks of metritis or abortion, the role of viruses in endometrial pathology and the mechanisms of viral infection of uterine cells are often ignored. The objectives of the present study were to explore the interaction, tropism and outcomes of BoHV-4 challenge of endometrial stromal and epithelial cells. Endometrial stromal and epithelial cells were purified and infected with a recombinant BoHV-4 carrying an enhanced green fluorescent protein (EGFP) expression cassette to monitor the establishment of infection. BoHV-4 efficiently infected both stromal and epithelial cells, causing a strong non-apoptotic cytopathic effect, associated with robust viral replication. The crucial step for the BoHV-4 endometriotropism appeared to be after viral entry as there was enhanced transactivation of the BoHV-4 immediate early 2 gene promoter following transient transfection into the endometrial cells. Infection with BoHV-4 increased cyclooxygenase 2 protein expression and prostaglandin estradiol secretion in endometrial stromal cells, but not epithelial cells. Bovine macrophages are persistently infected with BoHV-4, and co-culture with endometrial stromal cells reactivated BoHV-4 replication in the persistently infected macrophages, suggesting a symbiotic relationship between the cells and virus. In conclusion, the present study provides evidence of cellular and molecular mechanisms, supporting the concept that BoHV-4 is a pathogen associated with uterine disease.

Bacterial infection of endometrial stromal cells influences bovine herpesvirus 4 immediate early gene activation: a new insight into bacterial and viral interaction for uterine disease

Experimental infection with the gamma-herpesvirus bovine herpesvirus 4 (BoHV-4) rarely establishes disease, yet BoHV-4 is commonly associated with uterine disease in cattle. Uterine disease involves co-infection with bacteria such as Escherichia coli, which stimulate the production of prostaglandin E(2) (PGE(2)) by endometrial cells. BoHV-4 replication depends on immediate early 2 (IE2) gene transactivation and, in the present study, PGE(2), E. coli or its lipopolysaccharide upregulated the IE2 gene promoter in uterine cells. Bacterial co-infection is important for BoHV-4 uterine disease.

Potential of bovine herpesvirus 4 as a gene delivery vector.

A cloning system was developed for construction of BHV-4 recombinants and recombinant virus BHV-4EGFPDeltaTK containing an enhanced green fluorescent protein (EGFP) gene was constructed. The host range of BHV-4EGFPDeltaTK was characterized in vitro. When cell lines from various species and tissues were infected, most of the non-bovine cell lines exhibited neither cytopathic effect (CPE) nor supported viral replication, but EGFP expression was clearly observed. Next, embryonic stem cells were infected and induced to either non-specific or neural differentiation to determine whether they could survive and differentiate after BHV-4EGFPDeltaTK infection. Embryonic stem cells were infected successfully, as indicated by EGFP expression prior to differentiation, and EGFP expression could be detected in many differentiated cells. No CPE was noted. Therefore, BHV-4EGFPDeltaTK infection caused neither cell death nor interfered with non-specific or neural differentiation of embryonic stem cells. Finally, to assess the capability of BHV-4EGFPDeltaTK to infect post-mitotic neurons, cultures from brains of 2-weeks old mice were infected. No death of neuronal cells due to infection was observed and EGFP expression persisted for at least 15 days. Several biological characteristics of BHV-4 demonstrated previously make it a good candidate for a gene delivery vector. These include: little or no pathogenicity, unlikely oncogenicity, ability to establish persistent infection, and capability of herpesviruses to accommodate large amounts of foreign genetic material. These findings add the ability to infect several cell types coming from different animal species, usually without CPE, lack of interference with differentiation, and ability to maintain transgene expression in both undifferentiated and differentiated cells.

Detection of Mycobacterium Avium Subsp. Paratuberculosis in Bovine Fecal Samples: Comparison of Three Polymerase Chain Reaction—Based Diagnostic Tests with a Conventional Culture Method

Three commercially available assays, designed to specifically detect the presence of Mycobacterium avium subsp. paratuberculosis (MAP) in fecal samples by IS900-PCR, were compared with a conventional culture method. Fecal samples from 100 dairy cows were tested. Fifty-four (67.5%) of 80 culture-positive samples were positive for an assay that detects MAP DNA by dot spot hybridization of polymerase chain reaction products (kit A), 48 (60%) were positive by an assay using ethidium bromide staining for agar gel visualization of amplification products (kit B), and 49 (61.3%) were positive by an assay in which amplified products are detected by a colorimetric detection system (kit C). Relative sensitivity of all tests increased in proportion to the presence of MAP in fecal samples. Specificity was 100% based on results from 20 culture-negative samples from an MAP-free herd.

Association between Chlamydia psittaci seropositivity and abortion in Italian dairy cows

Although the seroprevalence of Chlamydia psittaci is widespread in Italian dairy herds, its role in inducing genital disorders has not been elucidated. We therefore set up a case-control study to compare seroprevalence to C. psittaci in an aborted-cow population and in a randomly selected control group in the province of Parma (the Po Valley of northern Italy). The true seroprevalence (45%) in aborted cows was significantly higher than that in the control group (24%) (adjusted odds ratio=2.53).

Potential Secondary Pathogenic Role for Bovine Herpesvirus 4

ABSTRACT Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with no clear disease association. Previous studies have demonstrated that macrophages can harbor persistent BoHV-4. We found that the addition of prostaglandin E2 (PGE2) to bovine macrophage cells persistently infected with BoHV-4 increases viral replication. Because opportunistic infection can increase PGE2 production, we propose a link between opportunistic infection, PGE2 production, and BoHV-4 replication.

Bovine endometrial stromal cells display osteogenic properties

The endometrium is central to mammalian fertility. The endometrial stromal cells are very dynamic, growing and differentiating throughout the estrous cycle and pregnancy. In humans, stromal cells appear to have progenitor or stem cell capabilities and the cells can even differentiate into bone. It is not clear whether bovine endometrial stromal cells exhibit a similar phenotypic plasticity. So, the present study tested the hypothesis that bovine endometrial stromal cells could be differentiated along an osteogenic lineage. Pure populations of bovine stromal cells were isolated from the endometrium. The endometrial stromal cell phenotype was confirmed by morphology, prostaglandin secretion, and susceptibility to viral infection. However, cultivation of the cells in standard endometrial cell culture medium lead to a mesenchymal phenotype similar to that of bovine bone marrow cells. Furthermore, the endometrial stromal cells developed signs of osteogenesis, such as alizarin positive nodules. When the stromal cells were cultured in a specific osteogenic medium the cells rapidly developed the characteristics of mineralized bone. In conclusion, the present study has identified that stromal cells from the bovine endometrium show a capability for phenotype plasticity similar to mesenchymal progenitor cells. These observations pave the way for further investigation of the mechanisms of stroma cell differentiation in the bovine reproductive tract.

Double immunization strategy with a BoHV-4-vectorialized secreted chimeric peptide BVDV-E2/BoHV-1-gD.

A bovine herpesvirus 4 was isolated from the milk cell fraction of a healthy cow and his full genome cloned as a bacterial artificial chromosome. So cloned viral genome was used as a vector platform to deliver in vitro and in vivo an optimized secreted chimeric peptide obtained by the fusion of the bovine viral diarrhoea virus glycoprotein E2 ectodomain with the bovine herpesvirus 1 glycoprotein D ectodomain. Recombinant virus infected cells robustly expressed and secreted the chimeric peptide into the culture medium and inoculated animals with the recombinant virus successfully responded toward antigens, gE2 and gD. Thus, this work has implications for the development of safe and effective polyvalent vaccines.

The Chemokine IL8 Is Up-Regulated in Bovine Endometrial Stromal Cells by the BoHV-4 IE2 Gene Product, ORF50/Rta: A Step Ahead Toward a Mechanism for BoHV-4 Induced Endometritis

Postpartum infections of the endometrium and metritis are common causes of delayed conception and infertility in cattle. These infections are characterized by inflammation of the endometrium and secretion of the chemokine interleukin 8 (IL8), which attracts granulocytes to the endometrium. Bovine herpesvirus 4 (BoHV-4) is tropic for the endometrium and the only virus consistently associated with postpartum metritis. The BoHV-4 Immediate Early 2 (IE2) gene is the first viral gene transcribed by host cells after infection, and the IE2 gene product, ORF50/Rta, transactivates host cell genes. The present study tested the hypothesis that ORF50/Rta transactivates the IL8 gene promoter during BoHV-4 infection of bovine endometrial stromal cells (BESCs). Infection of primary BESCs with BoHV-4 stimulated IL8 gene promoter activity and IL8 protein secretion. However, IL8 production was dependent on the transcription of viral genes, because psoralen/ultraviolet cross-linking of the viral DNA abrogated the response to BoHV-4 infection. Furthermore, IL8 promoter serial deletion analysis revealed a specific region responsive to ORF50/Rta. These observations may represent an endometrial defense mechanism against viral infection or a virulence mechanism by which viral replication stimulates chemokine secretion to attract more susceptible host cells to the endometrium.

Seroprevalence to bovine immunodeficiency virus and lack of association with leukocyte counts in Italian dairy cattle

We report herein on the first serological detection of antibodies to bovine immunodeficiency virus (BIV) in Italy. According to criteria of a stratified-random sampling of dairy cattle reared in the Parma area (a province in the Po Valley, Northern Italy), sera from 3166 cows belonging to 272 herds were collected. In addition, sera of 138 bulls from eight artificial-insemination (AI) centres were sampled. Seventy-eight cows (2.5%) from 16 herds (5.8%) and seven bulls (5.1%) from two AI centres were positive for BIV-R29 antibodies in the IFA-test. IFA-positive sera assayed by Western blot had reaction to different viral proteins: 81 out of 85 sera showed antibody to p26 (considered the BIV major internal core protein); four sera reacted to other viral proteins but not to p26. Peripheral blood leukocytes of 60 seropositive and 60 seronegative animals, belonging to eight BIV-infected herds, were enumerated to assess any effect of BIV infection on white-blood cells. No significant differences were detected between the two groups. These data indicate that BIV infection is present in Italian dairy cattle--but the role of BIV in inducing disease remains unclear.