Epizootic Hemorrhagic Disease Agar Gel Immunodiffusion Assay.

Agar gel immunodiffusion assay (AGID) is a laboratory test which detects specific antigen-antibody interactions by the development of visible precipitation lines in a semisolid matrix. Here we describe the preparation of agar gel plates, the method to test serum samples by AGID for the presence of EHDV antibodies, and the interpretation of test results. This test has known cross-reactivity to bluetongue antibodies; therefore positive samples by this assay require additional confirmatory testing; generally, its use should be limited to healthy animal attestations where required.

Immunogenicity and efficacy of Schmallenberg virus envelope glycoprotein subunit vaccines.

The Schmallenberg virus (SBV) is an orthobunyavirus that causes abortions, stillbirths, and congenital defects in pregnant sheep and cattle. Inactivated or live attenuated vaccines have been developed in endemic countries, but there is still interest in the development of SBV vaccines that would allow Differentiating Infected from Vaccinated Animals (DIVA). Therefore, an attempt was made to develop novel DIVA-compatible SBV vaccines using SBV glycoproteins expressed in baculovirus. All vaccines and phosphate buffered saline (PBS) controls were prepared with adjuvant and administered subcutaneously to cattle at 6 month of age. The first trial included 2 groups of animals vaccinated with either carboxyl-terminus glycoprotein (Gc) or PBS and boosted after 2 weeks. In the second trial, 3 groups of cattle were administered either Gc, Gc and amino-terminus glycoprotein (Gn), or PBS with a booster vaccination after 3 weeks. The animals were challenged with SBV 9 days after the booster vaccination in the first study, and 3 weeks after the booster vaccination in the second study. Using a SBV Gc-specific enzyme-linked immunosorbent assay, antibodies were first detected in serum samples 14 days after the first vaccination in both trials, and peaked on days 7 and 9 after the booster in the first and second trials, respectively. Low titers of neutralizing antibodies were detected in serum from only 3/6 and 2/4 animals in the first and second trial, respectively, at 14 days after the first vaccination. The titers increased 2 to 3-fold after the booster vaccination. SBV-specific RNA was detected in the serum and selective tissues in all animals after SBV challenge independent of vaccination status. The SBV candidate vaccines neither prevented viremia nor conferred protection against SBV infection.

Schmallenberg Disease-A Newly Emerged Culicoides-borne Viral Disease of Ruminants.

First appearing in 2011 in Northern Europe, Schmallenberg virus (SBV), an Orthobunyavirus of the Simbu serogroup, is associated with clinical disease mainly in ruminants such as cattle, sheep and goats. The clinical signs are characterized by abortion and congenital deformities in newborns. The virus is transmitted by Culicoides midges of the Obsoletus complex. SBV infection induces a solid protective immunity that persists for at least 4 or 6 years in sheep and cattle, respectively. SBV infection can be diagnosed directly by real-time RT-qPCR and virus isolation or indirectly by serological assays. Three vaccines are commercially available in Europe. This article provides a comprehensive literature review on this emerging disease regarding pathogenesis, transmission, diagnosis, control and prevention. This review also highlights that although much has been learned since SBV's first emergence, there are still areas that require further study to devise better mitigation strategies.

Virological and Serological Responses of Sheep and Cattle to Experimental Schmallenberg Virus Infection.

Schmallenberg virus (SBV) is an orthobunyavirus in the Simbu serogroup that emerged in Germany in late 2011 and was mostly associated with a mild transient disease of sheep and cattle. SBV is transmitted by biting midges (Culicoides species) and causes abortions, stillbirths, and congenital defects in naïve pregnant ruminants. Two separate studies were conducted with a primary objective of better understanding the virological and serological responses of sheep and cattle to different SBV isolates after experimental infection. The second objective was to produce immunoreagents and challenge materials for use in future vaccine and diagnostics research. These studies were carried out using the following infectious inocula: (i) infectious serum (IS) (ii) cell culture-grown virus, and (iii) infectious lamb brain homogenate. The responses were assessed in both species throughout the course of the experiment. SBV RNA in serum (RNAemia) was detected as early as 2 (in sheep) and 3 (in cattle) days postinfection (dpi) and peaked on 3 and 4 dpi in cattle and sheep, respectively. Cattle had higher levels of RNAemia compared with sheep. Experimental infection with IS resulted in the highest level of RNAemia in both species followed by cell culture-grown virus. A delayed, low level RNAemia was detected in cattle inoculated with infectious sheep brain. Isolation of SBV was only possible from 4 dpi sera from all cattle inoculated with IS and one sheep inoculated with cell culture-derived virus. SBV neutralizing antibodies were first detected on 14 dpi in both species. No specific gross and microscopic lesions were observed in either study. In conclusion, these studies highlight not only the difference in viremia and anti-SBV antibody level against the different SBV isolates, but also the extent of the response in the two host species.

Experimental Infection of Calves by Two Genetically-Distinct Strains of Rift Valley Fever Virus.

Recent outbreaks of Rift Valley fever in ruminant livestock, characterized by mass abortion and high mortality rates in neonates, have raised international interest in improving vaccine control strategies. Previously, we developed a reliable challenge model for sheep that improves the evaluation of existing and novel vaccines in sheep. This sheep model demonstrated differences in the pathogenesis of Rift Valley fever virus (RVFV) infection between two genetically-distinct wild-type strains of the virus, Saudi Arabia 2001 (SA01) and Kenya 2006 (Ken06). Here, we evaluated the pathogenicity of these two RVFV strains in mixed breed beef calves. There was a transient increase in rectal temperatures with both virus strains, but this clinical sign was less consistent than previously reported with sheep. Three of the five Ken06-infected animals had an early-onset viremia, one day post-infection (dpi), with viremia lasting at least three days. The same number of SA01-infected animals developed viremia at 2 dpi, but it only persisted through 3 dpi in one animal. The average virus titer for the SA01-infected calves was 1.6 logs less than for the Ken06-infected calves. Calves, inoculated with either strain, seroconverted by 5 dpi and showed time-dependent increases in their virus-neutralizing antibody titers. Consistent with the results obtained in the previous sheep study, elevated liver enzyme levels, more severe liver pathology and higher virus titers occurred with the Ken06 strain as compared to the SA01 strain. These results demonstrate the establishment of a virulent challenge model for vaccine evaluation in calves.

Inhibition of lung carcinogenesis and critical cancer-related signaling pathways by N-acetyl-S-(N-2-phenethylthiocarbamoyl)-l-cysteine, indole-3-carbinol and myo-inositol, alone and in combination.

In an extension of our earlier studies, we examined the inhibitory effects of N-acetyl-S-(N-2-phenethylthiocarbamoyl)-l-cysteine (PEITC-NAC), myo-inositol (MI) and indole-3-carbinol (I3C) or 3,3'-diindolylmethane (DIM), alone and in combination, on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) plus benzo[a]pyrene (BaP)-induced A/J mouse lung tumorigenesis and proliferation of A549 cells and human bronchial epithelial cells (HBECs) and relevant potential mechanisms. Mice treated with NNK plus BaP and fed non-supplemented diet had 13.0 + or - 4.1 lung tumors per mouse. Dietary feeding of mice with PEITC-NAC (5 mumol/g diet), I3C (5 mumol/g diet) or MI (56 mumol/g diet), beginning at 50% in the carcinogen treatment phase, significantly reduced tumor multiplicity to 8.2 + or - 2.0, 8.4 + or - 1.5 and 6.8 + or - 1.7 tumors per mouse, respectively. In mice given combinations of the chemopreventive agents, lung tumor multiplicity was significantly reduced to 6.3 + or - 2.2, 4.9 + or - 1.8, 4.8 + or - 1.9 and 3.6 + or - 1.4 by PEITC-NAC plus I3C, PEITC-NAC plus MI, I3C plus MI or PEITC-NAC plus I3C plus MI, respectively. Post-carcinogen administration of combinations of the agents also caused significant but weaker effects. Assessment of the anti-proliferative effects of the individual agents or their combinations showed significant reductions in the proliferation of cigarette smoke condensate (CSC)-pretreated HBEC (reduction by 30-41% at 48 h and 41-58% at 72 h) and A549 cells (30-43% at 48 h and 40-59% at 72 h), but not in dimethyl sulfoxide-pretreated HBEC. Combinatorial treatment with the agents also caused marked reductions in the activation of Akt, extracellular signal-regulated kinase and nuclear factor-kappaB in lung tumor tissues, CSC-pretreated HBEC and A549 cells. In conclusion, our studies demonstrated the promise of combinations of PEITC-NAC, I3C/DIM and MI for the chemoprevention of lung carcinogenesis in current and former smokers.