Association of FGF4L1 Retrogene Insertion with Prolapsed Gland of the Nictitans (Cherry Eye) in Dogs.

Cherry eye is the common name for prolapse of the nictitans gland, a tear-producing gland situated under the third eyelid of dogs. Cherry eye is characterized by a red fleshy protuberance in the corner of the eye, resembling a cherry. This protrusion is a displacement of the normal gland of the third eyelid, thought to be caused by a defect in the connective tissue that secures the gland in place. Options for treatment may include anti-inflammatory medications in mild cases, but surgical replacement of the gland is usually indicated. Cherry eye is most often seen in dogs under the age of two years, with certain breeds having a higher incidence, suggesting a potential genetic association. Integration of panel genetic testing into routine clinical practice allows for the generation of large numbers of genotyped individuals paired with clinical records and enables the investigation of common disorders using a genome-wide association study (GWAS) approach at scale. In this investigation, several thousand cases and controls for cherry eye in both purebred dogs and mixed breeds are used for a large-scale GWAS, revealing a single peak of genome-wide significance on canine chromosome 18, directly at the location of the previously identified FGF4 insertion known to cause chondrodysplasia in several breeds.

Missed, Not Missing: Phylogenomic Evidence for the Existence of Avian FoxP3.

The Forkhead box transcription factor FoxP3 is pivotal to the development and function of regulatory T cells (Tregs), which make a major contribution to peripheral tolerance. FoxP3 is believed to perform a regulatory role in all the vertebrate species in which it has been detected. The prevailing view is that FoxP3 is absent in birds and that avian Tregs rely on alternative developmental and suppressive pathways. Prompted by the automated annotation of foxp3 in the ground tit (Parus humilis) genome, we have questioned this assumption. Our analysis of all available avian genomes has revealed that the foxp3 locus is missing, incomplete or of poor quality in the relevant genomic assemblies for nearly all avian species. Nevertheless, in two species, the peregrine falcon (Falco peregrinus) and the saker falcon (F. cherrug), there is compelling evidence for the existence of exons showing synteny with foxp3 in the ground tit. A broader phylogenomic analysis has shown that FoxP3 sequences from these three species are similar to crocodilian sequences, the closest living relatives of birds. In both birds and crocodilians, we have also identified a highly proline-enriched region at the N terminus of FoxP3, a region previously identified only in mammals.

Cellular immunity in ASFV responses.

African swine fever virus (ASFV) infection usually results in an acute haemorrhagic disease with a mortality rate approaching 100% in domestic pigs. However, pigs can survive infection with less-virulent isolates of ASFV and may become chronically infected. Surviving animals are resistant to challenge with homologous or, in some cases, closely related isolates of the virus indicating that pigs can develop protective immunity against ASFV. During asymptomatic, non-virulent ASFV infections natural killer cell activity increases in pigs, suggesting this cell type plays a role in ASFV immunity. Furthermore, depletion of CD8(+) lymphocytes from ASFV immune pigs demolishes protective immunity against related virulent viruses. This suggests that ASFV specific antibody alone is not sufficient for protection against ASFV infection and that there is an important role for the CD8(+) lymphocyte subset in ASFV protective immunity. These results were supported by DNA immunization studies, demonstrating a correlation between the protection afforded against lethal challenge and the detection of a large number of vaccine-induced antigen-specific CD8(+) T-cells. Peripheral blood mononuclear cells (PBMCs) from ASF immune pigs protected from clinical disease show higher proportions of ASFV specific CD4(+)CD8(high+) double positive cytotoxic T cells than PBMCs from ASF immune but clinically diseased pig. The frequency of ASFV specific IFNγ producing T cells induced by immunization correlates to the degree of protection from ASFV challenge, and this may prove to be a useful indicator of any potential cross-protection against heterologous ASFV isolates.

Foot-and-mouth disease virus replicates only transiently in well-differentiated porcine nasal epithelial cells.

Three-dimensional (3D) porcine nasal mucosal and tracheal mucosal epithelial cell cultures were developed to analyze foot-and-mouth disease virus (FMDV) interactions with mucosal epithelial cells. The cells in these cultures differentiated and polarized until they closely resemble the epithelial layers seen in vivo. FMDV infected these cultures predominantly from the apical side, primarily by binding to integrin alphav beta6, in an Arg-Gly-Asp (RGD)-dependent manner. However, FMDV replicated only transiently without any visible cytopathic effect (CPE), and infectious progeny virus could be recovered only from the apical side. The infection induced the production of beta interferon (IFN-beta) and the IFN-inducible gene Mx1 mRNA, which coincided with the disappearance of viral RNA and progeny virus. The induction of IFN-beta mRNA correlated with the antiviral activity of the supernatants from both the apical and basolateral compartments. IFN-alpha mRNA was constitutively expressed in nasal mucosal epithelial cells in vitro and in vivo. In addition, FMDV infection induced interleukin 8 (IL-8) protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES mRNA in the infected epithelial cells, suggesting that it plays an important role in modulating the immune response.

Dramatic improvement in FMD DNA vaccine efficacy and cross-serotype antibody induction in pigs following a protein boost.

To overcome the low and slow development of humoral antibody often observed with DNA vaccines we applied a prime-boost strategy. When FMD DNA vaccine P1-2A3C3D and pGM-CSF primed pigs were boosted with inactivated foot-and-mouth disease virus (FMDV) antigen and recombinant 3D (without adjuvant) an average 36-fold increase in the FMDV antibody response was observed compared to conventional vaccination, that included a log(10) virus neutralising titre increase. Most remarkably, a significant level of cross-serotype reactivity was observed against A, C and Asia1 in the virus neutralisation and ELISA tests. This prime-boost strategy fully protected pigs from a heterologous challenge.

African swine fever virus causes microtubule-dependent dispersal of the trans-golgi network and slows delivery of membrane protein to the plasma membrane.

Viral interference with secretory cargo is a common mechanism for pathogen immune evasion. Selective down regulation of critical immune system molecules such as major histocompatibility complex (MHC) proteins enables pathogens to mask themselves from their host. African swine fever virus (ASFV) disrupts the trans-Golgi network (TGN) by altering the localization of TGN46, an organelle marker for the distal secretory pathway. Reorganization of membrane transport components may provide a mechanism whereby ASFV can disrupt the correct secretion and/or cell surface expression of host proteins. In the study reported here, we used the tsO45 temperature-sensitive mutant of the G protein of vesicular stomatitis virus to show that ASFV significantly reduces the rate at which the protein is delivered to the plasma membrane. This is linked to a general reorganization of the secretory pathway during infection and a specific, microtubule-dependent disruption of structural components of the TGN. Golgin p230 and TGN46 are separated into distinct vesicles, whereupon TGN46 is depleted. These data suggest that disruption of the TGN by ASFV can slow membrane traffic during viral infection. This may be functionally important because infection of macrophages with virulent isolates of ASFV increased the expression of MHC class I genes, but there was no parallel increase in MHC class I molecule delivery to the plasma membrane.

Identification of novel foot-and-mouth disease virus specific T-cell epitopes in c/c and d/d haplotype miniature swine.

To identify foot-and-mouth disease virus (FMDV) specific T-cell epitopes within the non-structural protein 3D in swine, pentadecapeptides were tested in proliferation and Interferon-gamma ELISPOT assays using lymphocytes from two strains of inbred miniature pigs (c/c and d/d haplotype) experimentally infected with FMDV. Lymphocytes of c/c pigs recognized peptides from three different regions in 3D, d/d lymphocytes recognized peptides from two regions, one of them being adjacent to an epitope of c/c pigs and comprising amino acid residues 346-370. Analyses of the response of d/d lymphocytes against peptides representing the structural protein 1A revealed another novel T-cell epitope. Investigation of the phenotype of responding lymphocytes showed a response of CD4(+)CD8(+)MHC-class-II(+) cells, identifying them as activated T-helper cells. This is the first report on FMDV specific T-cell epitopes recognized by swine leukocyte antigen (SLA) inbred swine and provides information useful for the design of novel vaccines against FMDV.

Perforin expression can define CD8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic T, natural killer T and MHC un-restricted cytotoxic T-cells.

In this study we have used the expression of perforin to characterize subsets of porcine cytotoxic lymphocytes. Perforin positive lymphocytes expressed both CD2 and CD8alpha, most were small dense lymphocytes (SDL) and up to 90% were CD3 negative. However, the numbers of perforin positive T-cells increased with the age of the animal and their populations increased after specific antigen stimulation in vitro. The remaining perforin positive lymphocytes were large and granular and contained more CD3+CD5+CD6+ T-cells (-40%) of which a substantial proportion also co-expressed CD4. Perforin was expressed in subpopulations of both CD8alphaalpha and CD8alphabeta lymphocytes, but was not expressed in gammadelta T-cells or monocyte/macrophages. The perforin positive CD3- subset was phenotypically homogeneous and defined as CD5-CD6-CD8beta-CD16+CD11b+. This population had NK activity and expressed mRNA for the NK receptor NKG2D, and adaptors DAP10 and DAP12. Perforin positive T-cells (CD3+) could be divided into at least three subsets. The first subset was CD4-CD5+CD6+CD11b-CD16- most were small dense lymphocytes with cytotoxic T-cell activity but not all expressed CD8beta. The second subset was mainly observed in the large granular lymphocytes. Their phenotype was CD4+CD5+CD6+CD8beta+CD16-CD11b- and also showed functional CTL activity. Thus not all of double positive T-cells are memory helper T-cells. The third subset did not express the T-cell co-receptor CD6, but up to half of them expressed another T-cell co-receptor CD5. The majority of this subset expressed CD11b and CD16, thus the third perforin positive T-cell subset was CD3+CD4-CD5+CD6-CD8beta+/-CD11b+CD16+, and possessed MHC-unrestricted cytotoxicity and LAK activity.