Psoroptes ovis-Early Immunoreactive Protein (Pso-EIP-1) a novel diagnostic antigen for sheep scab.

AIMS: Serodiagnosis of sheep scab is an established diagnostic method and has become popular in recent years. However, the current diagnostic antigen, Pso o 2, has shown promise as a component of a recombinant vaccine for scab, making it incompatible with discriminating between infected and vaccinated animals (DIVA). Here, we describe the discovery and characterization of a novel Psoroptes ovis immunodiagnostic antigen, P. ovis-Early Immunoreactive Protein-1 (Pso-EIP-1). METHODS AND RESULTS: Pso-EIP-1 is a highly abundant member of a six-gene family with no known homologs, indicating its potential uniqueness to P. ovis. Expression of recombinant Pso-EIP-1 (rPso-EIP-1) required a C-terminal fusion protein for stability and specific IgG immunoreactivity against rPso-EIP-1 was observed in sheep serum from 1 to 2 weeks post-infestation, indicating its highly immunogenic nature. Two of the three in silico-predicted B-cell epitopes of Pso-EIP-1 were confirmed by in vitro epitope mapping and, in a direct comparison by ELISA, Pso-EIP-1 performed to the same levels as Pso o 2 in terms of sensitivity, specificity and ability to diagnose P. ovis on sheep within 2 weeks of infestation. CONCLUSION: Pso-EIP-1 represents a novel diagnostic antigen for sheep scab with comparable levels of sensitivity and specificity to the existing Pso o 2 antigen.

Enhancing the toolbox to study IL-17A in cattle and sheep.

The development of methods to detect cytokine expression by T cell subsets in ruminants is fundamental to strategic development of new livestock vaccines for prevention of infectious diseases. It has been possible to detect T cell expression of IFN-γ, IL-4 and IL-10 in ruminants for many years but methods to detect expression of IL-17A are relatively limited. To address this gap in capability we have cloned bovine and ovine IL-17A cDNAs and expressed biologically-active recombinant proteins in Chinese Hamster Ovary (CHO) cells. We used the transfected CHO cells to screen commercially-available antibodies for their ability to detect IL-17A expression intracellularly and in culture supernates. We demonstrate that an ELISA for bovine IL-17A detects native ovine IL-17A. Moreover, the constituent polyclonal antibodies (pabs) in the ELISA were used to enumerate peripheral blood mononuclear cells (PBMC) expressing IL-17A from cattle and sheep by ELISpot. We identified two monoclonal antibodies (mabs) that detect recombinant intracellular IL-17A in CHO cells by flow cytometry. One of these mabs was used to detect native intracellular IL-17A expression in PBMC in conjunction with cell surface phenotyping mabs [CD4+ve, CD8+ve and Workshop Cluster 1 (WC-1)+ve gamma-delta (γδ)] we show that distinct T cell subsets in cattle (defined as CD4+ve, CD8+ve or WC-1+ve) and sheep (defined as CD4+ve or WC-1+ve) can express IL-17A following activation. These novel techniques provide a solid basis to investigate IL-17A expression and define specific CD4+ve T cell subset activation in ruminants.

A recombinant subunit vaccine for the control of ovine psoroptic mange (sheep scab).

Sheep scab, caused by infestation with the mite Psoroptes ovis, is highly contagious, causing intense pruritus and represents a major welfare and economic concern. Disease control strategies rely upon chemotherapy, however, sustainability is questionable due to issues of chemical residues, eco-toxicity and acaricide resistance. Control by vaccination is supported by demonstration of protective immunity in sheep previously infested with P. ovis. We identified vaccine candidates for P. ovis based on: (1) antigens selected by their interaction with host signalling pathways and the host immune-response; and (2) those shown to be either immunogenic or involved in mite feeding. This resulted in the development and validation, in repeated immunisation and challenge trials, of a seven recombinant protein sub-unit cocktail vaccine. Sheep were inoculated on three occasions, 2 weeks apart, along with QuilA adjuvant. Vaccination resulted in highly significant reductions in both lesion size (up to 63%) and mite numbers (up to 56%) following challenge. Mean lesion size in vaccinates was significantly smaller than controls from 1 week post infestation (wpi) until the end of the experiment at 6 wpi. All antigens elicited serum IgG responses following immunisation and prior to infestation, whereas controls did not produce antigen-specific IgG during the pre-infestation period. Vaccinated animals showed an amnestic response, with levels of antigen-specific IgG against muGST, Pso o 1 and Pso o 2 increasing following infestation. This vaccine represents the greatest reduction in lesion size to date with a sheep scab vaccine, providing encouragement for future production of a commercially-viable means of immunoprophylaxis.

Functional analysis of bovine TLR5 and association with IgA responses of cattle following systemic immunisation with H7 flagella.

Flagellin subunits are important inducers of host immune responses through activation of TLR5 when extracellular and the inflammasome if cytosolic. Our previous work demonstrated that systemic immunization of cattle with flagella generates systemic and mucosal IgA responses. The IgA response in mice is TLR5-dependent and TLR5 can impact on the general magnitude of the adaptive response. However, due to sequence differences between bovine and human/murine TLR5 sequences, it is not clear whether bovine TLR5 (bTLR5) is able to stimulate an inflammatory response following interaction with flagellin. To address this we have examined the innate responses of both human and bovine cells containing bTLR5 to H7 flagellin from E. coli O157:H7. Both HEK293 (human origin) and embryonic bovine lung (EBL) cells transfected with bTLR5 responded to addition of H7 flagellin compared to non-transfected controls. Responses were significantly reduced when mutations were introduced into the TLR5-binding regions of H7 flagellin, including an R90T substitution. In bovine primary macrophages, flagellin-stimulated CXCL8 mRNA and secreted protein levels were significantly reduced when TLR5 transcript levels were suppressed by specific siRNAs and stimulation was reduced with the R90T-H7 variant. While these results indicate that the bTLR5 sequence produces a functional flagellin-recognition receptor, cattle immunized with R90T-H7 flagella also demonstrated systemic IgA responses to the flagellin in comparison to adjuvant only controls. This presumably either reflects our findings that R90T-H7 still activates bTLR5, albeit with reduced efficiency compared to WT H7 flagellin, or that other flagellin recognition pathways may play a role in this mucosal response.

Characterisation of protective vaccine antigens from the thiol-containing components of excretory/secretory material of Ostertagia ostertagi.

Previous vaccination trials have demonstrated that thiol proteins affinity purified from Ostertagia ostertagi excretory-secretory products (O. ostertagi ES-thiol) are protective against homologous challenge. Here we have shown that protection induced by this vaccine was consistent across four independent vaccine-challenge experiments. Protection is associated with reduced cumulative faecal egg counts across the duration of the trials, relative to control animals. To better understand the diversity of antigens in O. ostertagi ES-thiol we used high-resolution shotgun proteomics to identify 490 unique proteins in the vaccine preparation. The most numerous ES-thiol proteins, with 91 proteins identified, belong to the sperm-coating protein/Tpx/antigen 5/pathogenesis-related protein 1 (SCP/TAPS) family. This family includes previously identified O. ostertagi vaccine antigens O. ostertagi ASP-1 and ASP-2. The ES-thiol fraction also has numerous proteinases, representing three distinct classes, including: metallo-; aspartyl- and cysteine proteinases. In terms of number of family members, the M12 astacin-like metalloproteinases, with 33 proteins, are the most abundant proteinase family in O. ostertagi ES-thiol. The O. ostertagi ES-thiol proteome provides a comprehensive database of proteins present in this vaccine preparation and will guide future vaccine antigen discovery projects.

The effect of Psoroptes ovis infestation on ovine epidermal barrier function.

Sheep scab is an intensively pruritic, exudative and allergic dermatitis of sheep caused by the ectoparasitic mite Psoroptes ovis. The purpose of the present study was to investigate the effect of P. ovis infestation on different components of the ovine epidermal barrier within the first 24 hours post-infestation (hpi). To achieve this, the expression of epidermal differentiation complex (EDC) genes and epidermal barrier proteins, the nature and severity of epidermal pathology and transepidermal water loss (TEWL) were evaluated.By 1 hpi a significant dermal polymorphonuclear infiltrate and a significant increase in TEWL with maximal mean TEWL (598.67 g/m2h) were observed. Epidermal pathology involving intra-epidermal pustulation, loss of epidermal architecture and damage to the basement membrane was seen by 3 hpi. Filaggrin and loricrin protein levels in the stratum corneum declined significantly in the first 24 hpi and qPCR validation confirmed the decrease in expression of the key EDC genes involucrin, filaggrin and loricrin observed by microarray analysis, with 5.8-fold, 4.5-fold and 80-fold decreases, respectively by 24 hpi.The present study has demonstrated that early P. ovis infestation disrupts the ovine epidermal barrier causing significant alterations in the expression of critical barrier components, epidermal pathology, and TEWL. Many of these features have also been documented in human and canine atopic dermatitis suggesting that sheep scab may provide a model for the elucidation of events occurring in the early phases of atopic sensitisation.

Suppression of ovine lymphocyte activation by Teladorsagia circumcincta larval excretory-secretory products.

Teladorsagia circumcincta is an important pathogenic nematode of sheep. It has been demonstrated previously that stimulation of murine T lymphocytes with excretory-secretory (ES) products derived from fourth stage larvae of T. circumcincta (Tci-L4-ES) results in de novo expression of Foxp3, a transcription factor intimately involved in regulatory T cell function. In the current study, Foxp3⁺ T cell responses in the abomasum and the effects of Tci-L4-ES on ovine peripheral blood mononuclear cells (PBMC) following T. circumcincta infection were investigated. T. circumcincta infection resulted in a significant increase in numbers of abomasal Foxp3⁺ T cells, but not an increase in the proportion of T cells expressing Foxp3. Unlike in mice, Tci-L4-ES was incapable of inducing T cell Foxp3 expression but instead suppressed mitogen-induced and antigen-specific activation and proliferation of ovine PBMC in vitro. This effect was heat labile, suggesting that it is mediated by protein(s). Suppression was associated with up-regulation of interleukin-10 (IL-10) mRNA, and specific monoclonal antibody neutralisation of IL-10 resulted in a 50% reduction in suppression, indicating involvement of the IL-10 signaling pathway. Suppression was significantly reduced in PBMC isolated from T. circumcincta infected vs. helminth-naïve lambs, and this reduction in suppression was associated with an increase in Tci-L4-ES antigen-specific T cells within the PBMC. In conclusion, we have identified a mechanism by which T. circumcincta may modulate the host adaptive immune response, potentially assisting survival of the parasite within the host. However, the impact of Tci-L4-ES-mediated lymphocyte suppression during T. circumcincta infection remains to be determined.

Efficacy of Phase I and Phase II Coxiella burnetii Bacterin Vaccines in a Pregnant Ewe Challenge Model.

The bacterium Coxiella burnetii can cause the disease Q-fever in a wide range of animal hosts. Ruminants, including sheep, are thought to play a pivotal role in the transmission of C. burnetii to humans; however, the only existing livestock vaccine, namely, Coxevac® (Ceva Animal Health Ltd., Libourne, France), a killed bacterin vaccine based on phase I C. burnetii strain Nine-Mile, is only approved for use in goats and cattle. In this study, a pregnant ewe challenge model was used to determine the protective effects of Coxevac® and an experimental bacterin vaccine based on phase II C. burnetii against C. burnetii challenge. Prior to mating, ewes (n = 20 per group) were vaccinated subcutaneously with either Coxevac®, the phase II vaccine, or were unvaccinated. A subset of pregnant ewes (n = 6) from each group was then challenged 151 days later (~100 days of gestation) with 106 infectious mouse doses of C. burnetii, Nine-Mile strain RSA493. Both vaccines provided protection against C. burnetii challenge as measured by reductions in bacterial shedding in faeces, milk and vaginal mucus, and reduced abnormal pregnancies, compared to unvaccinated controls. This work highlights that the phase I vaccine Coxevac® can protect ewes against C. burnetii infection. Furthermore, the phase II vaccine provided comparable levels of protection and may offer a safer and cost-effective alternative to the currently licensed vaccine.

Transcriptomic analysis of the temporal host response to skin infestation with the ectoparasitic mite Psoroptes ovis.

BACKGROUND: Infestation of ovine skin with the ectoparasitic mite Psoroptes ovis results in a rapid cutaneous immune response, leading to the crusted skin lesions characteristic of sheep scab. Little is known regarding the mechanisms by which such a profound inflammatory response is instigated and to identify novel vaccine and drug targets a better understanding of the host-parasite relationship is essential. The main objective of this study was to perform a combined network and pathway analysis of the in vivo skin response to infestation with P. ovis to gain a clearer understanding of the mechanisms and signalling pathways involved. RESULTS: Infestation with P. ovis resulted in differential expression of 1,552 genes over a 24 hour time course. Clustering by peak gene expression enabled classification of genes into temporally related groupings. Network and pathway analysis of clusters identified key signalling pathways involved in the host response to infestation. The analysis implicated a number of genes with roles in allergy and inflammation, including pro-inflammatory cytokines (IL1A, IL1B, IL6, IL8 and TNF) and factors involved in immune cell activation and recruitment (SELE, SELL, SELP, ICAM1, CSF2, CSF3, CCL2 and CXCL2). The analysis also highlighted the influence of the transcription factors NF-kB and AP-1 in the early pro-inflammatory response, and demonstrated a bias towards a Th2 type immune response. CONCLUSIONS: This study has provided novel insights into the signalling mechanisms leading to the development of a pro-inflammatory response in sheep scab, whilst providing crucial information regarding the nature of mite factors that may trigger this response. It has enabled the elucidation of the temporal patterns by which the immune system is regulated following exposure to P. ovis, providing novel insights into the mechanisms underlying lesion development. This study has improved our existing knowledge of the host response to P. ovis, including the identification of key parallels between sheep scab and other inflammatory skin disorders and the identification of potential targets for disease control.

The 1B vaccine strain of Chlamydia abortus produces placental pathology indistinguishable from a wild type infection.

Chlamydia abortus is one of the most commonly diagnosed causes of infectious abortion in small ruminants worldwide. Control of the disease (Enzootic Abortion of Ewes or EAE) is achieved using the commercial live, attenuated C. abortus 1B vaccine strain, which can be distinguished from virulent wild-type (wt) strains by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Published studies applying this typing method and whole-genome sequence analyses to cases of EAE in vaccinated and non-vaccinated animals have provided strong evidence that the 1B strain is not attenuated and can infect the placenta causing disease in some ewes. Therefore, the objective of this study was to characterise the lesions found in the placentas of ewes vaccinated with the 1B strain and to compare these to those resulting from a wt infection. A C. abortus-free flock of multiparous adult ewes was vaccinated twice, over three breeding seasons, each before mating, with the commercial C. abortus 1B vaccine strain (Cevac® Chlamydia, Ceva Animal Health Ltd.). In the second lambing season following vaccination, placentas (n = 117) were collected at parturition and analysed by C. abortus-specific real-time quantitative PCR (qPCR). Two placentas, from a single ewe, which gave birth to live twin lambs, were found to be positive by qPCR and viable organisms were recovered and identified as vaccine type (vt) by PCR-RFLP, with no evidence of any wt strain being present. All cotyledons from the vt-infected placentas were analysed by histopathology and immunohistochemistry and compared to those from wt-infected placentas. Both vt-infected placentas showed lesions typical of those found in a wt infection in terms of their severity, distribution, and associated intensity of antigen labelling. These results conclusively demonstrate that the 1B strain can infect the placenta, producing typical EAE placental lesions that are indistinguishable from those found in wt infected animals.

Characterisation of a niche-specific excretory-secretory peroxiredoxin from the parasitic nematode Teladorsagia circumcincta.

BACKGROUND: The primary cause of parasitic gastroenteritis in small ruminants in temperate regions is the brown stomach worm, Teladorsagia circumcincta. Host immunity to this parasite is slow to develop, consistent with the ability of T. circumcincta to suppress the host immune response. Previous studies have shown that infective fourth-stage T. circumcincta larvae produce excretory-secretory products that are able to modulate the host immune response. The objective of this study was to identify immune modulatory excretory-secretory proteins from populations of fourth-stage T. circumcincta larvae present in two different host-niches: those associated with the gastric glands (mucosal-dwelling larvae) and those either loosely associated with the mucosa or free-living in the lumen (lumen-dwelling larvae). RESULTS: In this study excretory-secretory proteins from mucosal-dwelling and lumen-dwelling T. circumcincta fourth stage larvae were analysed using comparative 2-dimensional gel electrophoresis. A total of 17 proteins were identified as differentially expressed, with 14 proteins unique to, or enriched in, the excretory-secretory proteins of mucosal-dwelling larvae. One of the identified proteins, unique to mucosal-dwelling larvae, was a putative peroxiredoxin (T. circumcincta peroxiredoxin 1, Tci-Prx1). Peroxiredoxin orthologs from the trematode parasites Schistosoma mansoni and Fasciola hepatica have previously been shown to alternatively activate macrophages and play a key role in promoting parasite induced Th2 type immunity. Here we demonstrate that Tci-Prx1 is expressed in all infective T. circumcincta life-stages and, when produced as a recombinant protein, has peroxidase activity, whereby hydrogen peroxide (H2O2) is reduced and detoxified. Furthermore, we use an in vitro macrophage stimulation assay to demonstrate that, unlike peroxiredoxins from trematode parasites Schistosoma mansoni and Fasciola hepatica, Tci-Prx1 is unable to alternatively activate murine macrophage cells. CONCLUSIONS: In this study, we identified differences in the excretory-secretory proteome of mucosal-dwelling and lumen-dwelling infective fourth-stage T. circumcincta larvae, and demonstrated the utility of this comparative proteomic approach to identify excretory-secretory proteins of potential importance for parasite survival and/or host immune modulation.

Niche-specific gene expression in a parasitic nematode; increased expression of immunomodulators in Teladorsagia circumcincta larvae derived from host mucosa.

Metazoan parasites have to survive in many different niches in order to complete their life-cycles. In the absence of reliable methods to manipulate parasite genomes and/or proteomes, identification of the molecules critical for parasite survival within these niches has largely depended on comparative transcriptomic and proteomic analyses of different developmental stages of the parasite; however, changes may reflect differences associated with transition between developmental stages rather than specific adaptations to a particular niche. In this study, we compared the transcriptome of two fourth-stage larval populations of the nematode parasite, Teladorsagia circumcincta, which were of the same developmental stage but differed in their location within the abomasum, being either mucosal-dwelling (MD) or lumen-dwelling (LD). Using RNAseq, we identified 57 transcripts which were significantly differentially expressed between MD and LD larvae. Of these transcripts, the majority (54/57) were up-regulated in MD larvae, one of which encoded for an ShKT-domain containing protein, Tck6, capable of modulating ovine T cell cytokine responses. Other differentially expressed transcripts included homologues of ASP-like proteins, proteases, or excretory-secretory proteins of unknown function. Our study demonstrates the utility of niche- rather than stage-specific analysis of parasite transcriptomes to identify parasite molecules of potential importance for survival within the host.

Possible mechanisms of host resistance to Haemonchus contortus infection in sheep breeds native to the Canary Islands.

Haemonchus contortus appears to be the most economically important helminth parasite for small ruminant production in many regions of the world. The two sheep breeds native to the Canary Islands display distinctly different resistant phenotypes under both natural and experimental infections. Canaria Hair Breed (CHB) tends to have significantly lower worm burden and delayed and reduced egg production than the susceptible Canaria Sheep (CS). To understand molecular mechanisms underlying host resistance, we compared the abomasal mucosal transcriptome of the two breeds in response to Haemonchus infection using RNAseq technology. The transcript abundance of 711 and 50 genes were significantly impacted by infection in CHB and CS, respectively (false discovery rate <0.05) while 27 of these genes were significantly affected in both breeds. Likewise, 477 and 16 Gene Ontology (GO) terms were significantly enriched in CHB and CS, respectively (P < 1.0 × 10(-4)). A broad range of mechanisms have evolved in resistant CHB to provide protection against the parasite. Our findings suggest that readily inducible acute inflammatory responses, complement activation, accelerated cell proliferation and subsequent tissue repair, and immunity directed against parasite fecundity all contributed to the development of host resistance to parasitic infection in the resistant breed.

A preliminary proteomic characterisation of extracellular vesicles released by the ovine parasitic nematode, Teladorsagia circumcincta.

Teladorsagia circumcincta is a major cause of ovine parasitic gastroenteritis in temperate climatic regions. The development of high levels of anthelmintic resistance in this nematode species challenges its future control. Recent research indicates that many parasite species release extracellular vesicles into their environment, many of which have been classified as endocytic in origin, termed exosomes. These vesicles are considered to play important roles in the intercellular communication between parasites and their hosts, and thus represent potentially useful targets for novel control strategies. Here, we demonstrate that exosome-like extracellular vesicles can be isolated from excretory-secretory (ES) products released by T. circumcincta fourth stage larvae (Tci-L4ES). Furthermore, we perform a comparative proteomic analysis of vesicle-enriched and vesicle-free Tci-L4ES. Approximately 73% of the proteins identified in the vesicle-enriched fraction were unique to this fraction, whilst the remaining 27% were present in both vesicle-enriched and vesicle-free fraction. These unique proteins included structural proteins, nuclear proteins, metabolic proteins, proteolytic enzymes and activation-associated secreted proteins. Finally, we demonstrate that molecules present within the vesicles-enriched material are targets of the IgA and IgG response in T. circumcincta infected sheep, and could potentially represent useful targets for future vaccine intervention studies.

Haplotype analysis of the PPARgamma Pro12Ala and C1431T variants reveals opposing associations with body weight.

BACKGROUND: Variation at the PPARG locus may influence susceptibility to type 2 diabetes and related traits. The Pro12Ala polymorphism may modulate receptor activity and is associated with protection from type 2 diabetes. However, there have been inconsistent reports of its association with obesity. The silent C1431T polymorphism has not been as extensively studied, but the rare T allele has also been inconsistently linked to increases in weight. Both rare alleles are in linkage disequilibrium and the independent associations of these two polymorphisms have not been addressed. RESULTS: We have genotyped a large population with type 2 diabetes (n = 1107), two populations of non-diabetics from Glasgow (n = 186) and Dundee (n = 254) and also a healthy group undergoing physical training (n = 148) and investigated the association of genotype with body mass index. This analysis has demonstrated that the Ala12 and T1431 alleles are present together in approximately 70% of the carriers. By considering the other 30% of individuals with haplotypes that only carry one of these polymorphisms, we have demonstrated that the Ala12 allele is consistently associated with a lower BMI, whilst the T1431 allele is consistently associated with higher BMI. CONCLUSION: This study has therefore revealed an opposing interaction of these polymorphisms, which may help to explain previous inconsistencies in the association of PPARG polymorphisms and body weight.

Transcriptomic analysis of circulating leukocytes reveals novel aspects of the host systemic inflammatory response to sheep scab mites.

Infestation of ovine skin with the ectoparasitic mite Psoroptes ovis results in the development of a rapid cutaneous inflammatory response, leading to the crusted skin lesions characteristic of sheep scab. To facilitate the identification of novel diagnostic and therapeutic targets, a better understanding of the host-parasite relationship in sheep scab is essential. Although our knowledge of the host's local cutaneous inflammatory response to sheep scab has increased in recent years, we still know relatively little about the mechanisms of this response at the systemic level. This study used a combined network and pathway analysis of the in vivo transcriptomic response of circulating leukocytes to infestation with P. ovis, during a 6 week period. Network graph analysis identified six temporally-associated gene clusters, which separated into two distinct sub-networks within the graph, representing those genes either up or down-regulated during the time course. Functional and pathway analysis of these clusters identified novel insights into the host systemic response to P. ovis infestation, including roles for the complement system, clotting cascade and fibrinolysis. These analyses also highlighted potential mechanisms by which the systemic immune response to sheep scab can influence local tissue responses via enhanced leukocyte activation and extravasation. By analysing the transcriptomic responses of circulating leukocytes in sheep following infestation with P. ovis, this study has provided key insights into the inflammatory response to infestation and has also demonstrated the utility of these cells as a proxy of events occurring at local tissue sites, providing insight into the mechanisms by which a local allergen-induced inflammatory response may be controlled.

The use of a Psoroptes ovis serodiagnostic test for the analysis of a natural outbreak of sheep scab.

BACKGROUND: Sheep scab is a highly contagious disease of sheep caused by the ectoparasitic mite Psoroptes ovis. The disease is endemic in the UK and has significant economic impact through its effects on performance and welfare. Diagnosis of sheep scab is achieved through observation of clinical signs e.g. itching, pruritis and wool loss and ultimately through the detection of mites in skin scrapings. Early stages of infestation are often difficult to diagnose and sub-clinical animals can be a major factor in disease spread. The development of a diagnostic assay would enable farmers and veterinarians to detect disease at an early stage, reducing the risk of developing clinical disease and limiting spread. METHODS: Serum samples were obtained from an outbreak of sheep scab within an experimental flock (n = 480 (3 samples each from 160 sheep)) allowing the assessment, by ELISA of sheep scab specific antibody prior to infestation, mid-outbreak (combined with clinical assessment) and post-treatment. RESULTS: Analysis of pre-infestation samples demonstrated low levels of potential false positives (3.8%). Of the 27 animals with clinical or behavioural signs of disease 25 tested positive at the mid-outbreak sampling period, however, the remaining 2 sheep tested positive at the subsequent sampling period. Clinical assessment revealed the absence of clinical or behavioural signs of disease in 132 sheep, whilst analysis of mid-outbreak samples showed that 105 of these clinically negative animals were serologically positive, representing potential sub-clinical infestations. CONCLUSIONS: This study demonstrates that this ELISA test can effectively diagnose sheep scab in a natural outbreak of disease, and more importantly, highlights its ability to detect sub-clinically infested animals. This ELISA, employing a single recombinant antigen, represents a major step forward in the diagnosis of sheep scab and may prove to be critical in any future control program.

Identification of CD4+CD25 high Foxp3+ T cells in ovine peripheral blood.

Regulatory T cells (Treg) are an important subset of T lymphocytes which play a key role in maintaining peripheral immunological tolerance. The most studied subpopulation of Treg in mice and humans are natural Treg, which differentiate in the thymus and are identified by expression of CD4, high levels of IL-2Rα (CD25), and forkhead box P3 (Foxp3), a transcription factor intimately associated with Treg function. We and others have previously identified Foxp3(+) T cells in ovine tissue, suggesting that Treg exist in this species. However, the existence of putative natural Treg in sheep, as identified by co-expression of CD4, CD25 and Foxp3, has yet to be determined. In this study we demonstrate that the anti-rat/mouse Foxp3 monoclonal antibody FJK-16s cross-reacts with ovine Foxp3. Using a transfected Chinese hamster ovary cell line that constitutively expresses recombinant ovine Foxp3 as a positive control, we have developed a sensitive triple-labelling flow cytometry protocol to simultaneously label CD4, CD25 and Foxp3. We demonstrate that Foxp3(+) T lymphocytes exist in ovine peripheral blood, and that the majority of Foxp3 expression occurs within the CD4(+)CD25(hi) population. These results are consistent with those seen in other mammalian species and indicate that putative natural Treg exist in sheep.

Diclofenac antagonizes peroxisome proliferator-activated receptor-gamma signaling.

Although nonsteroidal anti-inflammatory drugs (NSAIDs) are used as cancer chemopreventative agents, their mechanism is unclear because NSAIDs have cyclooxygenase-independent actions. We investigated an alternative target for NSAIDs, peroxisome proliferator-activated receptor-gamma (PPARgamma), activation of which decreases cancer cell proliferation. NSAIDs have been shown to activate this receptor, but only at high concentrations. Here, we have examined binding of diclofenac to PPARgamma using a cis-parinaric acid displacement assay and studied the effect of diclofenac effect on PPARgamma trans-activation in a COS-1 cell reporter assay. Unexpectedly, diclofenac bound PPARgamma at therapeutic concentrations (K(i) = 700 nM) but induced only 2-fold activation of PPARgamma at a concentration of 25 microM and antagonized PPARgamma trans-activation by rosiglitazone. This antagonism was overcome with increasing rosiglitazone concentrations, indicating that diclofenac is a partial agonist. No effect of diclofenac was seen without exogenous receptor, confirming that it was working through a PPARgamma-specific mechanism. This is the first description of an NSAID that can antagonize PPARgamma. In addition, this is the first time that an NSAID has been shown to bind this receptor at clinically meaningful concentrations. The physiological relevance of these findings was tested using adipocyte differentiation and cancer cell proliferation assays. Diclofenac decreased PPARgamma-mediated adipose cell differentiation by 60% and inhibited the action of rosiglitazone on the prostate cancer cell line, DU-145, allowing a 3-fold increase in proliferation. This work shows that standard doses of diclofenac may have pharmacodynamic interactions with rosiglitazone and this has therapeutic implications, both in the management of type 2 diabetes and during cancer treatment.