Equine enteroid-derived monolayers recapitulate key features of parasitic intestinal nematode infection.

Stem cell-derived organoid cultures have emerged as attractive experimental models for infection biology research regarding various types of gastro-intestinal pathogens and host species. However, the large size of infectious nematode larvae and the closed structure of 3-dimensional organoids often hinder studies of the natural route of infection. To enable easy administration to the apical surface of the epithelium, organoids from the equine small intestine, i.

Development of a 3-transcript host expression assay to differentiate between viral and bacterial infections in pigs.

Indiscriminate use of antibiotics to treat infections that are of viral origin contributes to unnecessary use which potentially may induce resistance in commensal bacteria. To counteract this a number of host gene transcriptional studies have been conducted to identify genes that are differently expressed during bacterial and viral infections in humans, and thus could be used as a tool to base decisions on the use of antibiotics. In this paper, we aimed to evaluate the potential of a selection of genes that have been considered biomarkers in humans, to differentially diagnose bacterial from viral infections in the pig.

Cytokine responses to various larval stages of equine strongyles and modulatory effects of the adjuvant G3 in vitro.

Aims: To generate different larval stages of Strongylus vulgaris and to study cytokine responses in cultures of eqPBMC exposed to defined larval stages of S. vulgaris and cyathostomins with the aim to understand the early immune reaction to these parasites.

Methods And Results: EqPBMC were exposed to S.

The adjuvant G3 promotes a Th1 polarizing innate immune response in equine PBMC.

The immunomodulatory effect of a new particulate adjuvant, G3, alone or in combination with agonists to TLR2/1 or TLR5 was evaluated in cultures of equine PBMC. Exposure to the G3 adjuvant up-regulated genes encoding IFN-γ, IL-1β, IL-6, IL-8, IL-12p40 and IL-23p19 in the majority of the horses tested, indicating that the G3 adjuvant induced a pro-inflammatory and Th1 dominated profile. In accordance, genes encoding IL-13, IL-4, IL-10 and TGF-β remained unaffected and genes encoding IFN-α, IL-17A and TNF-α were only occasionally and weakly induced.

A novel adjuvant G3 induces both Th1 and Th2 related immune responses in mice after immunization with a trivalent inactivated split-virion influenza vaccine.

A preferred adjuvant should promote both Th1 and Th2 responses. However, most adjuvants in common use are biased towards a Th2-driven response. Therefore, the ability of a novel saponin-based adjuvant G3 to inducing balanced Th1 and Th2 responses in BALB/c mice immunized with a split trivalent seasonal influenza vaccine was evaluated in comparison to that of the adjuvant Al(OH).

Innate immune responses induced by the saponin adjuvant Matrix-M in specific pathogen free pigs.

Saponin-based adjuvants have been widely used to enhance humoral and cellular immune responses in many species, but their mode of action is not fully understood. A characterization of the porcine transcriptional response to Matrix-M was performed in vitro using lymphocytes, monocytes or monocyte-derived dendritic cells (MoDCs) and in vivo. The effect of Matrix-M was also evaluated in specific pathogen free (SPF) pigs exposed to conventionally reared pigs.

Expression of T helper type 17 (Th17)-associated cytokines and toll-like receptor 4 and their correlation with Foxp3 positive cells in rectal biopsies of horses with clinical signs of inflammatory bowel disease.

Inflammatory bowel disease (IBD) in horses is an idiopathic disorder, encompassing different types of chronic intestinal inflammation. The pathogenesis of the disease remains to be established, but it has been suggested that an imbalance between regulatory T cells (Tregs) and T helper 17 (Th17)-associated cytokines and altered toll-like receptor 4 (TLR4) expression is associated with intestinal inflammation in other species. The aim of the present study was to quantify Tregs in rectal biopsies from horses affected with IBD by immunohistochemistry and to evaluate expression of genes encoding interleukin (IL)-12p40, IL-17A, IL-23p19 and TLR4 by real-time quantitative PCR.

PCV2 on the spot-A new method for the detection of single porcine circovirus type 2 secreting cells.

A porcine circovirus type 2 SPOT (PCV2-SPOT) assay was established to enumerate virus-secreting lymphocytes obtained from naturally infected pigs. The assay is based on the same principle as general ELISPOT assays but instead of detecting cytokine or immunoglobulin secretion, PCV2 particles are immobilized and detected as filter spots. The method was used to evaluate the influence of various cell activators on the PCV2 secretion in vitro and was also applied to study the PCV2 secretion by lymphocytes obtained from pigs in healthy herds and in a herd afflicted by postweaning multisystemic wasting disease (PMWS).

Early inflammatory response to the saponin adjuvant Matrix-M in the pig.

The early inflammatory response to Matrix-M was evaluated in pigs. Adverse reactions measured as body temperature, appetite, activity level and reaction at the site of injection were not observed after s.c.

Expression of reference genes and T helper 17 associated cytokine genes in the equine intestinal tract.

There is accumulating evidence for the involvement of pro-inflammatory cytokines associated with a T helper 17 response in intestinal disorders such as inflammatory bowel disease (IBD) in humans. The involvement of interleukin (IL)-17 or IL-23 in equine IBD has not been studied and most gene expression studies in the equine intestine have been limited to the use of a single non-validated reference gene. In this study, expression of the reference gene candidates β2 microglobulin (β2M), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone H2A type 1, hypoxanthine-guanine phosphoribosyltransferase (HPRT), 60S ribosomal protein L32 (RPL32), succinate dehydrogenase complex subunit A (SDHA) and transferrin receptor 1 protein coding (TFRC)in the equine intestine was evaluated by quantitative PCR.

Expression of tlr4, md2 and cd14 in equine blood leukocytes during endotoxin infusion and in intestinal tissues from healthy horses.

The expression of tlr4, md2 and cd14 was studied in equine blood leukocytes and in intestinal samples using real time PCR. The stability of three commonly used reference genes, glyceraldehyde-3P-dehydrogenase (GAPDH), hypoxantine ribosyltransferase (HPRT) and succinate dehydrogenase complex subunit A (SDHA), was evaluated using qbase(PLUS). The equine peripheral blood mononuclear cells (eqPBMC) examined were either stimulated in vitro with Phorbol 12-myristate 13-acetate (PMA) and ionomycin or with the CpG oligodeoxynuclotide 2216 (CpG-ODN 2216) or obtained from horses before, during and after infusion of endotoxin.

Isolation in cell cultures and initial characterisation of two novel bocavirus species from swine in Northern Ireland.

We report the isolation in cell cultures of two novel bocavirus species in pigs from farms in Northern Ireland with clinical postweaning multisystemic wasting syndrome (PMWS). We have designated the isolates as porcine bocavirus-3 (PBoV3) and porcine bocavirus-4 (PBoV4). To date 5082 and 4125 bps of PBoV3 and PBoV4 have been sequenced, respectively.

Development and comparison of a Primer-Probe Energy Transfer based assay and a 5' conjugated Minor Groove Binder assay for sensitive real-time PCR detection of infectious laryngotracheitis virus.

In this study the design and development of two real-time PCR assays for the rapid, sensitive and specific detection of infectious laryngotracheitis virus (ILTV) DNA is described. A Primer-Probe Energy Transfer (PriProET) assay and 5' conjugated Minor Groove Binder (MGB) method are compared and contrasted. Both have been designed to target the thymidine kinase gene of the ILTV genome.

Pan-serotypic detection of foot-and-mouth disease virus using a minor groove binder probe reverse transcription polymerase chain reaction assay.

A novel assay for the pan-serotypic detection of foot-and-mouth disease virus (FMDV) was designed using a 5' conjugated minor groove binder (MGB) probe real-time RT-PCR system. This assay targets the 3D region of the FMDV genome and is capable of detecting 20 copies of a transcribed RNA standard. The linear range of the test was eight logs from 2 × 10¹ to 2 × 10⁸ copies and amplification time was approximately 2 h.

Development of a minor groove binder assay for real-time one-step RT-PCR detection of swine vesicular disease virus.

The design and development of a 5' conjugated minor groove binder (MGB) probe real-time RT-PCR assay are described for rapid, sensitive and specific detection of swine vesicular disease virus (SVDV) RNA. The assay is designed to target the 2C gene of the SVDV genome and is capable of detecting 2×10(2) copies of an RNA standard per reaction. It does not detect any of the other RNA viruses that cause vesicular disease in pigs, or the human enterovirus, Coxsackie B5 virus (CVB5) which is closely related antigenically to SVDV.

Sensitive detection of African swine fever virus using real-time PCR with a 5' conjugated minor groove binder probe.

The design of a 5' conjugated minor groove binder (MGB) probe real-time PCR assay is described for the rapid, sensitive and specific detection of African swine fever virus (ASFV) DNA. The assay is designed against the 9GL region and is capable of detecting 20 copies of a DNA standard. It does not detect any of the other common swine DNA viruses tested in this study.

Molecular beacon real-time PCR detection of swine viruses.

Rapid and reliable detection of viral pathogens is critical for the management of the diseases threatening the economic competitiveness of the swine farming industry worldwide. Molecular beacon assays are one type of real-time polymerase chain reaction (PCR) technology capable of fast, specific, sensitive, and reliable viral detection. In this paper, the development of molecular beacon assays as novel tools for the rapid detection of Aujeszky's disease virus, African swine fever virus, porcine circovirus type 2 and porcine parvovirus is described.

Adaptation of an Invader assay for the detection of African swine fever virus DNA.

A closed tube isothermal Invader assay (Third Wave Technologies Inc., Madison, Wisconsin, USA) was adapted for the detection of African swine fever virus (ASFV) DNA. Several ASFV Invader assays were designed successfully and tested on a real-time PCR instrument (iCycler, BioRad).

Interaction of classical swine fever virus with dendritic cells.

Functional disruption of dendritic cells (DCs) is an important strategy for viral pathogens to evade host defences. Monocytotropic viruses such as classical swine fever virus (CSFV) could employ such a mechanism, since the virus can suppress immune responses and induce apoptosis without infecting lymphocytes. Here, CSFV was shown to infect and efficiently replicate in monocyte- and in bone marrow-derived DCs.

Both the P and V proteins of the porcine rubulavirus LPMV interact with the NP protein via their respective C-terminal unique parts.

In this paper we show that the porcine rubulavirus LPMV phosphoprotein (P) and V protein (V) both interact with the nucleoprotein (NP). There are also indications for an interaction between P and V with L protein. Further analysis of the domains of the P and V which are necessary for interaction with the NP protein demonstrates that the interaction is not mediated from their common part but instead from their unique C-terminal parts, respectively.

Porcine rubulavirus LPMV RNA persists in the central nervous system of pigs after recovery from acute infection.

In order to study persistence of the porcine rubulavirus LPMV, we examined tissue samples collected from pigs 53 days after experimental infection. These pigs survived the initial infection and could clinically be considered to have recovered from the infection. Two of the pigs used in this study were chemically immunosuppressed during the last 4 days before necropsy.

Multiple factors including subgenomic RNAs and reduced viral protein expression are associated with a persistent infection by porcine rubulavirus (LPMV).

The synthesis of virus specific RNA and the expression of viral proteins in PK-15 cells persistently infected with the porcine rubulavirus LPMV have been studied at two different cell-passages following establishment of persistency (passages 25 and 65). Protein analysis of persistently infected cells and the virus particles released from these failed to demonstrate the presence of the polymerase (L) protein. A decrease in the amount of the phospho- (P) protein was also noted.

Establishment and characterisation of a porcine rubulavirus (LPMV) persistent infection in porcine kidney cells.

Porcine rubulavirus (LPMV) can establish persistent infections in porcine kidney cells. Cell cultures characterised at passages 25 and 65 demonstrated haemadsorption, formation of syncytia, and a slower growth rate. The nucleoprotein (NP) and haemagglutinin-neuraminidase (HN) protein were present in all cells, although not to the same extent as in wild type infected cells.

The molecular biology of the porcine paramyxovirus LPMV.

Protein and genomic studies of a previously uncharacterized porcine paramyxovirus (designated LPMV) confirmed that it was a member of the paramyxovirus genus. The nucleotide sequences and deduced amino acids of the complete P-gene, M-gene, F-gene and HN-gene as well as the intergenic sequences have been determined. Comparative sequence analysis of the M-gene of LPMV revealed the closest relationship of LPMV was to human mumps virus with a homology of 46% and 55% at the amino acid and nucleotide levels respectively.

The P gene of the porcine paramyxovirus LPMV encodes three possible polypeptides P, V and C: the P protein mRNA is edited.

The nucleotide sequence of the P gene of the porcine paramyxovirus La-Piedad-Michoacan-Mexico virus (LPMV) was analysed. Three long open reading frames (ORFs) were found in the mRNA sense. Insertion of two G residues is necessary to obtain an ORF encoding the P protein, which gives a P protein of 404 amino acids with a calculated Mr of 42475.