Comparison of potential nuclear precursors for prolactin and growth hormone messenger RNA

Recombinant DNA plasmids containing the coding sequence for rat prolactin or rat growth hormone have been used to investigate the presence of possible precursors for prolactin and growth hormone mRNA. Cytoplasmic and nuclear RNA was prepared from either rat pituitaries or fromthe GC pituitary cell line. RNA was electrophoresed on agarose gels containing methylmercury hydroxide and then transferred to diazobenzyloxymethyl paper. The paper was then hybridized to prolactin or growth hormone recombinant DNA probes labeled in vitro with 32P. The prolactin probe hybridized to RNA species of 7.0, 6.4, 3.8, 1.7, and 1.0 kilobases in nuclear RNA and only to a 1.0-kilobase species in cytoplasmic RNA. Hybridization with a growth hormone probe demonstrated nuclear RNA species of 6.7, 5.6, 2.3, and 1.0 kilobases. These findings demonstrate the presence of multiple species of prolactin and growth hormone RNA which are larger larger than the mature cytoplasmic mRNAs. The large nuclear RNAs are likely precursors for prolactin and growth hormone mRNA.     

Protection of IFNAR (?/?) Mice against Bluetongue Virus Serotype 8, by Heterologous (DNA/rMVA) and Homologous (rMVA/rMVA) Vaccination,Expressing Outer-Capsid Protein VP2

The protective efficacy of recombinant vaccines expressing serotype 8 bluetongue virus (BTV-8) capsid proteins was tested in a mouse model. The recombinant vaccines comprised plasmid DNA or Modified Vaccinia Ankara viruses encoding BTV VP2, VP5 or VP7 proteins. These constructs were administered alone or in combination using either a homologous prime boost vaccination regime (rMVA/rMVA) or a heterologous vaccination regime (DNA/rMVA). The DNA/rMVA or rMVA/rMVA prime-boost were administered at a three week interval and all of the animals that received VP2 generated neutralising antibodies. The vaccinated and non-vaccinated-control mice were subsequently challenged with a lethal dose of BTV-8. Mice vaccinated with VP7 alone were not protected. However, mice vaccinated with DNA/rMVA or rMVA/rMVA expressing VP2, VP5 and VP7 or VP2 alone were all protected.     

Bovine plasmacytoid dendritic cells are the major source of type I interferon in response to foot-and-mouth disease virus in vitro and in vivo

Type I interferons (alpha/beta interferons [IFN-α/β]) are the main innate cytokines that are able to induce a cellular antiviral state, thereby limiting viral replication and disease pathology. Plasmacytoid dendritic cells (pDCs) play a crucial role in the control of viral infections, especially in response to viruses that have evolved mechanisms to block the type I IFN signal transduction pathway. Using density gradient separation and cell sorting, we have highly enriched a population of bovine cells capable of producing high levels of biologically active type I IFN. These cells represented less than 0.1% of the total lymphocyte population in blood, pseudoafferent lymph, and lymph nodes. Phenotypic analysis identified these cells as bovine pDCs (CD3(-) CD14(-) CD21(-) CD11c(-) NK(-) TCRδ(-) CD4(+) MHC II(+) CD45RB(+) CD172a(+) CD32(+)). High levels of type I IFN were generated by these cells in vitro in response to Toll-like receptor 9 (TLR-9) agonist CpG and foot-and-mouth disease virus (FMDV) immune complexes. In contrast, immune complexes formed with UV-inactivated FMDV or FMDV empty capsids failed to elicit a type I IFN response. Depletion of CD4 cells in vivo resulted in levels of type I IFN in serum early during FMDV infection that were significantly lower than those for control animals. In conclusion, pDCs interacting with immune-complexed virus are the major source of type I interferon production during acute FMDV infection in cattle.     

Potential for epidemic take-off from the primary outbreak farm via livestock movements

Background

We consider the potential for infection to spread in a farm population from the primary outbreak farm via livestock movements prior to disease detection. We analyse how this depends on the time of the year infection occurs, the species transmitting, the length of infectious period on the primary outbreak farm, location of the primary outbreak, and whether a livestock market becomes involved. We consider short infectious periods of 1 week, 2 weeks and 4 weeks, characteristic of acute contagious livestock diseases. The analysis is based on farms in Scotland from 1 January 2003 to 31 July 2007.

Results

The proportion of primary outbreaks from which an acute contagious disease would spread via movement of livestock is generally low, but exhibits distinct annual cyclicity with peaks in May and August. The distance that livestock are moved varies similarly: at the time of the year when the potential for spread via movements is highest, the geographical spread via movements is largest. The seasonal patterns for cattle differ from those for sheep whilst there is no obvious seasonality for pigs. When spread via movements does occur, there is a high risk of infection reaching a livestock market; infection of markets can amplify disease spread. The proportion of primary outbreaks that would spread infection via livestock movements varies significantly between geographical regions.

Conclusions

In this paper we introduce a set-up for analysis of movement data that allows for a generalized assessment of the risk associated with infection spreading from a primary outbreak farm via livestock movements, applying this to Scotland, we assess how this risk depends upon the time of the year, species transmitting, location of the farm and other factors.     

The canine isolate Lactobacillus acidophilus LAB20 adheres to intestinal epithelium and attenuates LPS-induced IL-8 secretion of enterocytes in vitro

Background

For a good probiotic candidate, the abilities to adhere to intestinal epithelium and to fortify barrier function are considered to be crucial for colonization and functionality of the strain. The strain Lactobacillus acidophilus LAB20 was isolated from the jejunum of a healthy dog, where it was found to be the most pre-dominant lactobacilli. In this study, the adhesion ability of LAB20 to intestinal epithelial cell (IECs) lines, IECs isolated from canine intestinal biopsies, and to canine, porcine and human intestinal mucus was investigated. Further, we studied the ability of LAB20 to fortify the epithelial cell monolayer and to reduce LPS-induced interleukin (IL-8) release from enterocytes.

Results

We found that LAB20 presented higher adhesion to canine colonic mucus as compared to mucus isolated from porcine colon. LAB20 showed adhesion to HT-29 and Caco-2 cell lines, and importantly also to canine IECs isolated from canine intestinal biopsies. In addition, LAB20 increased the transepithelial electrical resistance (TER) of enterocyte monolayers and thus strengthened the intestinal barrier function. The strain showed also anti-inflammatory capacity in being able to attenuate the LPS-induced IL-8 production of HT-29 cells.

Conclusion

In conclusion, canine indigenous strain LAB20 is a potential probiotic candidate for dogs adhering to the host epithelium and showing intestinal barrier fortifying and anti-inflammatory effects.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-014-0337-9) contains supplementary material, which is available to authorized users.     

Unexpected photobleaching of Alexa 488 in a fixed bacterial sample during 2-photon excitation

A sample of fixed bacterial cells was examined by immunofluorescence microscopy using an Alexa 488 conjugated secondary antibody for visualization. Excitation using visible light confirmed the expected photostability of this fluorophore; however, when using 2-photon excitation, Alexa 488 was rapidly and substantially photobleached. The unexpected instability of Alexa 488 under certain conditions may have deleterious consequences if not anticipated and accommodated in experimental protocols.     

Associations between lamb survival and prion protein genotype: analysis of data for ten sheep breeds in Great Britain

Background  

Control of brucellosis in livestock, wildlife and humans depends on the reliability of the methods used for detection and identification of bacteria. In the present study, we describe the evaluation of the recently established real-time PCR assay based on the Brucella-specific insertion sequence IS711 with blood samples from 199 wild boars (first group of animals) and tissue samples from 53 wild boars (second group of animals) collected in Switzerland. Results from IS711 real-time PCR were compared to those obtained by bacterial isolation, Rose Bengal Test (RBT), competitive ELISA (c-ELISA) and indirect ELISA (i-ELISA).