The effect of neonatal vaccination for bovine respiratory disease in the face of a dual challenge with bovine viral diarrhea virus and Mannheimia hemolytica.

Bovine respiratory disease is the greatest threat to calf health. In this study, colostrum-fed dairy X beef calves were vaccinated at ∼30 days of age with an adjuvanted parenteral vaccine containing modified live bovine viral diarrhea virus (BVDV) type 1 and type 2, bovine herpesvirus 1 (BHV-1), bovine parainfluenza type 3 virus (PI3V) and bovine respiratory syncytial virus (BRSV) andM. haemolyticatoxoid (Group 1), or intranasal temperature-sensitive BHV-1, BRSV and PI3V concurrently witha parenteral vaccine containing modified live BVDV type 1 and type 2 andM. haemolyticatoxoid (Group 2) or a placebo (Group 3). The calves were challenged ∼150 days post vaccination intranasally with BVDV 1b and then 7 days later intratracheally withM. haemolytica. The calves wereeuthanized 6 days after theM. haemolyticachallenge. Clinical signs following BVDV infection were similar in all groups. There was increased rectal temperatures in the Groups 2 and 3 on day 3 and in Group 3 on days 8-13. Group 1 animals had a slight leukopenia following BVDV infection while Groups 2 and 3 had greater leukopenia. BVDV type 1 and 2 serum titers increased in Group 1 following vaccination while these titers waned in Groups 2 and 3. There were higher levels of BVDV in the buffy coats and nasal samples in Group 2 and Group 3 versus Group 1 (p < 0.01). Interferon-gamma response was higher (p < 0.01) in Group 1 animals than Groups 2 and 3. Group 1 had the lowest percent pneumonic tissue (1.6%) while Group 2 vaccinates had 3.7% and the control Group 3 was 5.3%. Vaccination in the face of maternal antibody with a parenteral adjuvanted vaccine resulted in better protection than the regimen of an intranasal vaccine anda parenteral adjuvanted BVDV andM haemolyticacombination vaccine in a BVDV-M. haemolyticadual challenge.

Efficacy of oleandrin and PBI-05204 against bovine viruses of importance to commercial cattle health.

BACKGROUND: Bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV). and bovine coronavirus (BCV) threaten the productivity of cattle worldwide. Development of therapeutics that can control the spread of these viruses is an unmet need. The present research was designed to explore the in vitro antiviral activity of the Nerium oleander derived cardiac glycoside oleandrin and a defined N. oleander plant extract (PBI-05204) containing oleandrin. METHODS: Madin Darby Bovine Kidney (MDBK) cells, Bovine Turbinate (BT) cells, and Human Rectal Tumor-18 (HRT-18) cells were used as in vitro culture systems for BVDV, BRSV and BCV, respectively. Cytotoxicity was established using serial dilutions of oleandrin or PBI-05204. Noncytotoxic concentrations of each drug were used either prior to or at 12 h and 24 h following virus exposure to corresponding viruses. Infectious virus titers were determined following each treatment. RESULTS: Both oleandrin as well as PBI-05204 demonstrated strong antiviral activity against BVDV, BRSV, and BCV, in a dose-dependent manner, when added prior to or following infection of host cells. Determination of viral loads by PCR demonstrated a concentration dependent decline in virus replication. Importantly, the relative ability of virus produced from treated cultures to infect new host cells was reduced by as much as 10,000-fold at noncytotoxic concentrations of oleandrin or PBI-05204. CONCLUSIONS: The research demonstrates the potency of oleandrin and PBI-05204 to inhibit infectivity of three important enveloped bovine viruses in vitro. These data showing non-toxic concentrations of oleandrin inhibiting infectivity of three bovine viruses support further investigation of in vivo antiviral efficacy.

Characterization of bovine ileal epithelial cell line for lectin binding, susceptibility to enteric pathogens, and TLR mediated immune responses.

In this study, primary and immortalized bovine intestinal epithelial cells (BIECs) were characterized for the expression of surface carbohydrate moieties. Primary BIEC-c4 cells showed staining greater than 90 % for 16 lectins but less than 50 % staining for four lectins. Immortalized BIECs showed significantly different lectin binding profile for few lectins compared to BIEC-c4 cells. BIEC-c4 cells were studied for infectivity to E. coli, Salmonella enterica, bovine rotavirus, bovine coronavirus, and bovine viral diarrhea virus. Bovine strain E. coli B41 adhered to BIEC-c4 cells and Salmonella strains S. Dublin and S. Mbandaka showed strong cell invasion. BIEC-c4 cells were susceptible to bovine rotavirus. LPS stimulation upregulated IL-10, IL-8, and IL-6 expression and Poly I:C upregulated TLR 8 and TLR 9 expression. This study provides important knowledge on the glycoconjugate expression profile of primary and immortalized BIECs and infectivity and immune responses of primary BIECs to bacterial and viral pathogens or ligands.

The Effect of Bovine Viral Diarrhea Virus (BVDV) Strains and the Corresponding Infected-Macrophages' Supernatant on Macrophage Inflammatory Function and Lymphocyte Apoptosis.

Bovine viral diarrhea virus (BVDV) is an important viral disease of cattle that causes immune dysfunction. Macrophages are the key cells for the initiation of the innate immunity and play an important role in viral pathogenesis. In this in vitro study, we studied the effect of the supernatant of BVDV-infected macrophage on immune dysfunction. We infected bovine monocyte-derived macrophages (MDM) with high or low virulence strains of BVDV. The supernatant recovered from BVDV-infected MDM was used to examine the functional activity and surface marker expression of normal macrophages as well as lymphocyte apoptosis. Supernatants from the highly virulent 1373-infected MDM reduced phagocytosis, bactericidal activity and downregulated MHC II and CD14 expression of macrophages. Supernatants from 1373-infected MDM induced apoptosis in MDBK cells, lymphocytes or BL-3 cells. By protein electrophoresis, several protein bands were unique for high-virulence, 1373-infected MDM supernatant. There was no significant difference in the apoptosis-related cytokine mRNA (IL-1beta, IL-6 and TNF-a) of infected MDM. These data suggest that BVDV has an indirect negative effect on macrophage functions that is strain-specific. Further studies are required to determine the identity and mechanism of action of these virulence factors present in the supernatant of the infected macrophages.

A novel bovine papillomavirus type in the genus Dyokappapapillomavirus.

Papillomaviruses are a diverse group of viruses that are known to infect a wide range of animal species. Bovine papillomaviruses (BPVs) are divided into at least 21 genotypes (BPV1 to BPV21),  with most BPV isolates/strains described to date belonging to one of four genera, including Deltapapillomavirus, Xipapillomavirus, Epsilonpapillomavirus and Dyoxipapillomavirus. Here, we describe the identification and genetic characterization of a new BPV type in the genus Dyokappapapillomavirus. A farm in the state of New York, USA, reported chronic cases of vulvovaginitis in Holstein cows in 2016. Biopsies and/or swab samples collected from the vaginal mucosa were subjected to diagnostic investigation. Conventional diagnostic assays yielded negative results, and vaginal swab samples were subjected to viral metagenomic sequencing. Notably, BLAST searches revealed a papillomavirus genome with 7480 bp in length (67% nt sequence identity to BPV16). Additionally, phylogenetic analysis of the L1 gene of the papillomavirus identified here (tentatively named BPV22) revealed that it clusters with members of the genus Dyokappapapillomavirus. Interestingly, the recently identified BPV16, which was detected in fibropapilloma lesions in cattle also clusters within the Dyokappapapillomavirus group. Each virus, however, forms a separate branch in the phylogenetic tree. These results indicate that the putative BPV22 represents the second BPV within the genus Dyokappapapillomavirus.

Priming Cross-Protective Bovine Viral Diarrhea Virus-Specific Immunity Using Live-Vectored Mosaic Antigens.

Bovine viral diarrhea virus (BVDV) plays a key role in bovine respiratory disease complex, which can lead to pneumonia, diarrhea and death of calves. Current vaccines are not very effective due, in part, to immunosuppressive traits and failure to induce broad protection. There are diverse BVDV strains and thus, current vaccines contain representative genotype 1 and 2 viruses (BVDV-1 & 2) to broaden coverage. BVDV modified live virus (MLV) vaccines are superior to killed virus vaccines, but they are susceptible to neutralization and complement-mediated destruction triggered by passively acquired antibodies, thus limiting their efficacy. We generated three novel mosaic polypeptide chimeras, designated NproE2123; NS231; and NS232, which incorporate protective determinants that are highly conserved among BVDV-1a, 1b, and BVDV-2 genotypes. In addition, strain-specific protective antigens from disparate BVDV strains were included to broaden coverage. We confirmed that adenovirus constructs expressing these antigens were strongly recognized by monoclonal antibodies, polyclonal sera, and IFN-γ-secreting T cells generated against diverse BVDV strains. In a proof-of-concept efficacy study, the multi-antigen proto-type vaccine induced higher, but not significantly different, IFN-γ spot forming cells and T-cell proliferation compared to a commercial MLV vaccine. In regards to the humoral response, the prototype vaccine induced higher BVDV-1 specific neutralizing antibody titers, whereas the MLV vaccine induced higher BVDV-2 specific neutralizing antibody titers. Following BVDV type 2a (1373) challenge, calves immunized with the proto-type or the MLV vaccine had lower clinical scores compared to naïve controls. These results support the hypothesis that a broadly protective subunit vaccine can be generated using mosaic polypeptides that incorporate rationally selected and validated protective determinants from diverse BVDV strains. Furthermore, regarding biosafety of using a live vector in cattle, we showed that recombinant human adenovirus-5 was cleared within one week following intradermal inoculation.

The effects of vaccination on serum hormone concentrations and conception rates in synchronized naive beef heifers.

Crossbred beef heifers (N = 59) were vaccinated at the time of synchronization/breeding with either a commercially available bovine herpesvirus type 1 modified live virus (MLV) (one dose) or inactivated virus vaccine (one or two doses). The estrus cycle was synchronized at vaccination and heifers were artificially inseminated 8 days (one dose) or 36 days (two dose) after initial vaccination. Pregnancy rates were greater for control heifers (90%; P = 0.02) and heifers given the inactivated virus vaccine (one dose: 86%; P = 0.08; or two: 90%; P < 0.01) than those given the MLV vaccine (48%). No control heifers experienced an abnormal estrous cycle, whereas only two (two dose; 2/21) and one (one dose; 1/7) heifers in the inactive virus groups had abnormal estrous cycles and were similar to control (P > 0.10). Heifers given the MLV vaccine had a greater (P = 0.02) percentage of abnormal estrous cycles (38%; 8/21) compared with the control and inactivated groups. Of the heifers with an abnormal estrous cycle, 100% of heifers given the inactivated vaccine (one or two dose) conceived at their return estrus, whereas only 38% of heifers given the MLV vaccine conceived at their return estrus (P > 0.10). During the synchronization period, concentrations of estrogen were greater (P < 0.01) in the control and the two-dose inactivated group compared with the MLV group. After AI, progesterone concentrations were greater (P < 0.01) in control heifers compared with the inactivated and MLV groups, but were similar (P ≥ 0.18) between the inactivated and MLV groups. Therefore, naïve heifers vaccinated with the inactivated vaccine were less likely to have an abnormal estrous cycle and had significantly higher pregnancy rates compared with heifers vaccinated with the MLV vaccine. In summary, vaccination of naïve heifers with an MLV vaccine at the start of a fixed-time AI protocol had a negative effect on pregnancy success.

Proliferative activity of Echinacea angustifolia root extracts on cancer cells: Interference with doxorubicin cytotoxicity.

Doxorubicin is an anticancer drug that causes apoptosis in cells, but cardiotoxicity limits the cumulative dose that can remain in the blood. Echinacea extracts have been prescribed to supplement cancer chemotherapy. In a recent study, it was reported that Echinacea purpurea extracts protected noncancerous cells from apoptosis. Our study aimed to determine interference with doxorubicin chemotherapy, and if fractions and compounds from Echinacea angustifolia roots protected the cells. Cervical and breast cancer cells were treated with the Echinacea samples and doxorubicin. At 0.05 and 0.5 microM doxorubicin concentration, cynarine increased HeLa cell growth by 48-125% and 29-101%, respectively (p<0.01). At 0.05 microM doxorubicin concentration, chicoric acid increased cell growth by 23-100% (p<0.01). When MCF-7 cells were treated with Echinacea and doxorubicin, the ethyl acetate fraction increased cell growth by 20-25%, and chicoric acid increased cell growth by 10-15%. Cynarine showed proliferative activity on HeLa cells, but showed antiproliferative activity on MCF-7 cells. Results indicate that phenolic compounds are responsible for proliferative activity. Studies with individual compounds show that chicoric acid and cynarine interfered with cells treated with 0.5 microM doxorubicin. The results of this study show that Echinacea herbal medicines affect cell proliferation despite cancer treatment, and that herbal medicines require further study with respect to anticancer drugs.

The immune response to bovine viral diarrhea virus: a constantly changing picture.

Bovine viral diarrhea virus (BVDV) is one of the major immuno-suppressive viruses of cattle. The effect on the innate and acquired immune system is unique and results in dramatic immune dysfunction. BVDV infection also has the ability to cause persistent infection (PI) in the developing fetus. This Pl syndrome creates a requirement for high levels of BVDV immunity from vaccines to prevent these infections. BVDV vaccines and their future development continue to be an enigma in the control of BVDV.

Detection of bovine leukemia virus in blood and milk by nested and real-time polymerase chain reactions.

Concerns about retroviruses in livestock and products derived from them have necessitated the development of tests to detect the bovine leukemia virus (BLV) in blood and milk from cattle. Dairy cattle (n = 101) from 5 different geographical areas were used for this study. A nested polymerase chain reaction (PCR) identified 98% of BLV seropositive cattle (n = 80) from blood and 65% from milk, whereas real-time PCR detected 94% of BLV seropositive cattle from blood and 59% from milk. Bovine leukemia virus was also detected by PCR in approximately 10% of seronegative cattle (n = 21), most likely because of early detection before seroconversion.

Effect of genistein on replication of bovine herpesvirus type 1.

OBJECTIVE: To study the antiviral activity of genistein, a soya isoflavone, on in vitro replication of bovine herpesvirus type 1 (BHV-1). SAMPLE POPULATION: Madin-Darby bovine kidney (MDBK) cells. PROCEDURE: Effects of genistein on the magnitude and kinetics of inhibition of BHV-1 phosphorylation of glycoprotein E (gE) and in vitro replication of BHV-1 in MDBK cells were evaluated. Antiviral activity of genistein was compared with 2 compounds, estradiol-17beta (EST) and tamoxifen (TAM), that have estrogenic and antiestrogenic activity, respectively. High-performance liquid chromatography (HPLC) was used to determine the concentration of genistein in medium from infected and uninfected MDBK cultures. RESULTS: Genistein reduced BHV-1, but not gE-deleted BHV-1 (BHV-1gEdelta3.1), replication by 90% at 18 hours after inoculation. This inhibition was not sustained through 24 hours after inoculation. The genistein concentration in media from MDBK cells was decreased by 40% during BHV-1 infection, compared with 16% for uninfected cells, at 24 hours after inoculation. Genistein inhibited gE phosphorylation and BHV-1 replication in a dose-dependent manner. Dosing with 25 microM genistein at 0 and 12 hours after inoculation of BHV-1 was optimal for decreasing BHV-1 replication. Estradiol-17beta EST and TAM did not affect BHV-1 replication. CONCLUSIONS AND CLINICAL RELEVANCE: The decrease in genistein concentration was a viral infection-dependent event. Genistein is an inhibitor of BHV-1 replication because of its ability to inhibit tyrosine kinase activity. A possible application may be for the control of BHV-1 infection in cattle by feeding soya products rich in genistein prior to or during periods of stress.