Field evaluation of poultry red mite (Dermanyssus gallinae) native and recombinant prototype vaccines.

Vaccination is a desirable emerging strategy to combat poultry red mite (PRM), Dermanyssus gallinae. We performed trials, in laying hens in a commercial-style cage facility, to test the vaccine efficacy of a native preparation of soluble mite extract (SME) and of a recombinant antigen cocktail vaccine containing bacterially-expressed versions of the immunogenic SME proteins Deg-SRP-1, Deg-VIT-1 and Deg-PUF-1. Hens (n=384 per group) were injected with either vaccine or adjuvant only (control group) at 12 and 17 weeks of age and then challenged with PRM 10days later. PRM counts were monitored and, at the termination of the challenge period (17 weeks post challenge), average PRM counts in cages containing birds vaccinated with SME were reduced by 78% (p<0.001), compared with those in the adjuvant-only control group. When the trial was repeated using the recombinant antigen cocktail vaccine, no statistically significant differences in mean PRM numbers were observed in cages containing vaccinated or adjuvant-only immunised birds. The roles of antigen-specific antibody levels and duration in providing vaccine-induced and exposure-related protective immunity are discussed.

Characterisation of tropomyosin and paramyosin as vaccine candidate molecules for the poultry red mite, Dermanyssus gallinae.

BACKGROUND: Dermanyssus gallinae is the most economically important haematophagous ectoparasite in commercial egg laying flocks worldwide. It infests the hens during the night where it causes irritation leading to restlessness, pecking and in extreme cases anaemia and increased cannibalism. Due to an increase in the occurrence of acaricide-resistant D. gallinae populations, new control strategies are required and vaccination may offer a sustainable alternative to acaricides. In this study, recombinant forms of D. gallinae tropomyosin (Der g 10) and paramyosin (Der g 11) were produced, characterised and tested as vaccine candidate molecules. METHODS: The D. gallinae paramyosin (Der g 11) coding sequence was characterised and recombinant versions of Der g 11 and D. gallinae tropomyosin (Der g 10) were produced. Hens were immunised with the recombinant proteins and the resulting antibodies were fed to D. gallinae and mite mortality evaluated. Sections of mites were probed with anti- Der g 11 and Der g 10 antibodies to identify the tissue distribution of these protein in D. gallinae. RESULTS: The entire coding sequence of Der g 11 was 2,622 bp encoding 874 amino acid residues. Immunohistochemical staining of mite sections revealed that Der g 10 and Der g 11 were located throughout D. gallinae tissues. In phylogenetic analyses of these proteins both clustered with orthologues from tick species rather than with orthologues from astigmatid mites. Antibodies raised in hens against recombinant forms of these proteins significantly increased D. gallinae mortality, by 19 % for Der g 10 (P < 0.001) and by 23 % for Der g 11 (P = 0.009) when fed to the mites using an in vitro feeding device. CONCLUSIONS: This study has shown that Der g 10 and Der g 11 were located ubiquitously throughout D. gallinae and that antibodies raised against recombinant versions of these proteins can be used to significantly increase D. gallinae mortality in an in vitro feeding assay. When comparing archived data for all recombinant and native proteins assessed as vaccines using this in vitro feeding assay, Der g 10 and Der g 11 ranked highly and performed better than some of the pools of native proteins.

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.

Identification and evaluation of vaccine candidate antigens from the poultry red mite (Dermanyssus gallinae).

An aqueous extract of the haematophagous poultry ectoparasite, Dermanyssus gallinae, was subfractionated using anion exchange chromatography. Six of these subfractions were used to immunise hens and the blood from these hens was fed, in vitro, to poultry red mites. Mite mortality following these feeds was indicative of protective antigens in two of the subfractions, with the risks of mites dying being 3.1 and 3.7 times higher than in the control group (P<0.001). A combination of two-dimensional immunoblotting and immunoaffinity chromatography, using IgY from hens immunised with these subfractions, was used in concert with proteomic analyses to identify the strongest immunogenic proteins in each of these subfractions. Ten of the immunoreactive proteins were selected for assessment as vaccine candidates using the following criteria: intensity of immune recognition; likelihood of exposure of the antigen to the antibodies in a blood meal; proposed function and known vaccine potential of orthologous molecules. Recombinant versions of each of these 10 proteins were produced in Escherichia coli and were used to immunise hens. Subsequent in vitro feeding of mites on blood from these birds indicated that immunisation with Deg-SRP-1 (serpin), Deg-VIT-1 (vitellogenin), Deg-HGP-1 (hemelipoglycoprotein) or Deg-PUF-1 (a protein of unknown function) resulted in significantly increased risk of mite death (1.7-2.8times higher than in mites fed blood from control hens immunised with adjuvant only, P<0.001). The potential for using these antigens in a recombinant vaccine is discussed.

Characterisation of Dermanyssus gallinae glutathione S-transferases and their potential as acaricide detoxification proteins.

BACKGROUND: Glutathione S-transferases (GSTs) facilitate detoxification of drugs by catalysing the conjugation of the reduced glutathione (GSH) to electrophilic xenobiotic substrates and therefore have a function in multi-drug resistance. As a result, knowledge of GSTs can inform both drug resistance in, and novel interventions for, the control of endo- and ectoparasite species. Acaricide resistance and the need for novel control methods are both pressing needs for Dermanyssus gallinae, a highly economically important haematophagous ectoparasite of poultry. METHODS: A transcriptomic database representing D. gallinae was examined and 11 contig sequences were identified with GST BlastX identities. The transcripts represented by 3 contigs, designated Deg-GST-1, -2 and -3, were fully sequenced and further characterized by phylogenetic analysis. Recombinant versions of Deg-GST-1, -2 and -3 (rDeg-GST) were enzymically active and acaricide-binding properties of the rDeg-GSTs were established by evaluating the ability of selected acaricides to inhibit the enzymatic activity of rDeg-GSTs. RESULTS: 6 of the identified GSTs belonged to the mu class, followed by 3 kappa, 1 omega and 1 delta class molecules. Deg-GST-1 and -3 clearly partitioned with orthologous mu class GSTs and Deg-GST-2 partitioned with delta class GSTs. Phoxim, permethrin and abamectin significantly inhibited rDeg-GST-1 activity by 56, 35 and 17% respectively. Phoxim also inhibited rDeg-2-GST (14.8%) and rDeg-GST-3 (20.6%) activities. CONCLUSIONS: Deg-GSTs may have important roles in the detoxification of pesticides and, with the increased occurrence of acaricide resistance in this species worldwide, Deg-GSTs are attractive targets for novel interventions.

Optimizing the Protection of Cattle against Escherichia coli O157:H7 Colonization through Immunization with Different Combinations of H7 Flagellin, Tir, Intimin-531 or EspA.

Enterohemorrhagic Escherichia coli (EHEC) are important human pathogens, causing hemorrhagic colitis and hemolytic uraemic syndrome in humans. E. coli O157:H7 is the most common serotype associated with EHEC infections worldwide, although other non-O157 serotypes cause life-threatening infections. Cattle are a main reservoir of EHEC and intervention strategies aimed at limiting EHEC excretion from cattle are predicted to lower the risk of human infection. We have previously shown that immunization of calves with recombinant versions of the type III secretion system (T3SS)-associated proteins EspA, intimin and Tir from EHEC O157:H7 significantly reduced shedding of EHEC O157 from experimentally-colonized calves, and that protection could be augmented by the addition of H7 flagellin to the vaccine formulation. The main aim of the present study was to optimize our current EHEC O157 subunit vaccine formulations by identifying the key combinations of these antigens required for protection. A secondary aim was to determine if vaccine-induced antibody responses exhibited cross-reactive potential with antigens from other EHEC serotypes. Immunization with EspA, intimin and Tir resulted in a reduction in mean EHEC O157 shedding following challenge, but not the mean proportion of calves colonized. Removal of Tir resulted in more prolonged shedding compared with all other groups, whereas replacement of Tir with H7 flagellin resulted in the highest levels of protection, both in terms of reducing both mean EHEC O157 shedding and the proportion of colonized calves. Immunization of calves with recombinant EHEC O157 EspA, intimin and Tir resulted in the generation of antibodies capable of cross-reacting with antigens from non-O157 EHEC serotypes, suggesting that immunization with these antigens may provide a degree of cross-protection against other EHEC serotypes. Further studies are now required to test the efficacy of these vaccines in the field, and to formally test the cross-protective potential of the vaccines against other non-O157 EHEC.

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.

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.

Development of a cDNA microarray for the measurement of gene expression in the sheep scab mite Psoroptes ovis.

BACKGROUND: Sheep scab is caused by the ectoparasitic mite Psoroptes ovis which initiates a profound cutaneous inflammatory response, leading to the development of the skin lesions which are characteristic of the disease. Existing control strategies rely upon injectable endectocides and acaricidal dips but concerns over residues, eco-toxicity and the development of acaricide resistance limit the sustainability of this approach. In order to identify alternative means of disease control, a deeper understanding of both the parasite and its interaction with the host are required. METHODS: Herein we describe the development and utilisation of an annotated P. ovis cDNA microarray containing 3,456 elements for the measurement of gene expression in this economically important ectoparasite. The array consists of 981 P. ovis EST sequences printed in triplicate along with 513 control elements. Array performance was validated through the analysis of gene expression differences between fed and starved P. ovis mites. RESULTS: Sequences represented on the array include homologues of major house dust mite allergens and tick salivary proteins, along with factors potentially involved in mite reproduction and xenobiotic metabolism. In order to validate the performance of this unique resource under biological conditions we used the array to analyse gene expression differences between fed and starved P. ovis mites. These analyses identified a number of house dust mite allergen homologues up-regulated in fed mites and P. ovis transcripts involved in stress responses, autophagy and chemosensory perception up-regulated in starved mites. CONCLUSION: The P. ovis cDNA microarray described here has been shown to be both robust and reproducible and will enable future studies to analyse gene expression in this important ectoparasite.

Assessment of cathepsin D and L-like proteinases of poultry red mite, Dermanyssus gallinae (De Geer), as potential vaccine antigens.

Vaccination is a feasible strategy for controlling the haematophagous poultry red mite Dermanyssus gallinae. A cDNA library enriched for genes upregulated after feeding was created to identify potential vaccine antigens. From this library, a gene (Dg-CatD-1) encoding a 383 amino acid protein (Dg-CatD-1) with homology to cathepsin D lysosomal aspartyl proteinases was identified as a potential vaccine candidate. A second gene (Dg-CatL-1) encoding a 341 amino acid protein (Dg-CatL-1) with homology to cathepsin L cysteine proteinases was also selected for further study. IgY obtained from naturally infested hens failed to detect Dg-CatD-1 suggesting that it is a concealed antigen. Conversely, Dg-CatL-1 was detected by IgY derived from natural-infestation, indicating that infested hens are exposed to Dg-CatL-1. Mortality rates 120 h after mites had been fed anti-Dg-CatD-1 were significantly higher than those fed control IgY (PF<0·01). In a survival analysis, fitting a proportional hazards model to the time of death of mites, anti-Dg-CatD-1 and anti-Dg-CatL-1 IgY had 4·42 and 2·13 times higher risks of dying compared with controls (PF<0·05). Dg-CatD-1 and L-1 both have potential as vaccine antigens as part of a multi-component vaccine and have the potential to be improved as vaccine antigens using alternative expression systems.

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.

Recent developments in the diagnosis of ectoparasite infections and disease through a better understanding of parasite biology and host responses.

Some conventional methods of diagnosis of ectoparasite infections can have low sensitivity and/or specificity. In addition, early infestations, sub-clinical and carrier hosts often go un-diagnosed, allowing infestations to spread. This review focuses on the important ectoparasites of human, livestock and companion animals for which improved diagnostic tools are either already in use, or in development. These advances in diagnostic technologies have resulted in improved treatment, control and preventative strategies for many ectoparasitic diseases. Immunodiagnostic methods have had a large impact, with the emergence of highly sensitive and specific enzyme-linked immunosorbent assays (ELISAs) for sarcoptic and psoroptic mange, with further improved tests in development. In the present review, the advantages and limitations of such tests are discussed and the potential for future development explored. The increasing use of molecular tools, for example, PCR and other molecular methods, has improved our understanding of the epidemiology of ectoparasitic diseases, with practical consequences for community-based control programmes. Recently, the identification of specific signalling pathways during the host response to ectoparasites has led to the identification of disease biomarkers which, along with new technologies, such as multiplexed assays and microfluidic platforms, could lead to more cost-effective, rapid and accurate diagnosis of infectious diseases.

Development of a serodiagnostic test for sheep scab using recombinant protein Pso o 2.

Early stages of sheep scab, the disease caused by the non-burrowing mite Psoroptes ovis, are often sub-clinical, or can be mis-diagnosed. A diagnostic test capable of detecting early disease and latent infestations is therefore highly desirable in disease control. This paper describes the design and validation of an ELISA, which incorporates a recombinant P. ovis antigen (Pso o 2), for the early detection of anti-P. ovis serum antibodies in sheep. This ELISA was evaluated using sera from sheep infested with P. ovis (n = 58) and sheep (n = 433) with no P. ovis infestation as well as sheep infected with other parasites including gastrointestinal nematodes (GIN), or chewing lice. A receiver operating characteristic (ROC) curve analysis was generated using the ELISA results for 491 sheep sera with the area under the curve (AUC) being 0.97. An optimal OD(450) cut-off of >0.06 absorbance units gave a test sensitivity of 0.93 and specificity of 0.90. The Pso o 2-based ELISA was able to detect specific antibodies to P. ovis during early experimental infestation prior to disease patency, indicating its utility for detecting sub-clinical infestation.

Host transcription factors in the immediate pro-inflammatory response to the parasitic mite Psoroptes ovis.

BACKGROUND: Sheep scab, caused by infestation with the ectoparasitic mite Psoroptes ovis, results in the rapid development of cutaneous inflammation and leads to the crusted skin lesions characteristic of the disease. We described previously the global host transcriptional response to infestation with P. ovis, elucidating elements of the inflammatory processes which lead to the development of a rapid and profound immune response. However, the mechanisms by which this response is instigated remain unclear. To identify novel methods of intervention a better understanding of the early events involved in triggering the immune response is essential. The objective of this study was to gain a clearer understanding of the mechanisms and signaling pathways involved in the instigation of the immediate pro-inflammatory response. RESULTS: Through a combination of transcription factor binding site enrichment and pathway analysis we identified key roles for a number of transcription factors in the instigation of cutaneous inflammation. In particular, defined roles were elucidated for the transcription factors NF-kB and AP-1 in the orchestration of the early pro-inflammatory response, with these factors being implicated in the activation of a suite of inflammatory mediators. CONCLUSIONS: Interrogation of the host temporal response to P. ovis infestation has enabled the further identification of the mechanisms underlying the development of the immediate host pro-inflammatory response. This response involves key regulatory roles for the transcription factors NF-kB and AP-1. Pathway analysis demonstrated that the activation of these transcription factors may be triggered following a host LPS-type response, potentially involving TLR4-signalling and also lead to the intriguing possibility that this could be triggered by a P. ovis allergen.

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.

Generation, analysis and functional annotation of expressed sequence tags from the ectoparasitic mite Psoroptes ovis.

BACKGROUND: Sheep scab is caused by Psoroptes ovis and is arguably the most important ectoparasitic disease affecting sheep in the UK. The disease is highly contagious and causes and considerable pruritis and irritation and is therefore a major welfare concern. Current methods of treatment are unsustainable and in order to elucidate novel methods of disease control a more comprehensive understanding of the parasite is required. To date, no full genomic DNA sequence or large scale transcript datasets are available and prior to this study only 484 P. ovis expressed sequence tags (ESTs) were accessible in public databases. RESULTS: In order to further expand upon the transcriptomic coverage of P. ovis thus facilitating novel insights into the mite biology we undertook a larger scale EST approach, incorporating newly generated and previously described P. ovis transcript data and representing the largest collection of P. ovis ESTs to date. We sequenced 1,574 ESTs and assembled these along with 484 previously generated P. ovis ESTs, which resulted in the identification of 1,545 unique P. ovis sequences. BLASTX searches identified 961 ESTs with significant hits (E-value < 1E-04) and 584 novel P. ovis ESTs. Gene Ontology (GO) analysis allowed the functional annotation of 880 ESTs and included predictions of signal peptide and transmembrane domains; allowing the identification of potential P. ovis excreted/secreted factors, and mapping of metabolic pathways. CONCLUSIONS: This dataset currently represents the largest collection of P. ovis ESTs, all of which are publicly available in the GenBank EST database (dbEST) (accession numbers FR748230 - FR749648). Functional analysis of this dataset identified important homologues, including house dust mite allergens and tick salivary factors. These findings offer new insights into the underlying biology of P. ovis, facilitating further investigations into mite biology and the identification of novel methods of intervention.

Genome-wide transcriptomic analysis of intestinal tissue to assess the impact of nutrition and a secondary nematode challenge in lactating rats.

BACKGROUND: Gastrointestinal nematode infection is a major challenge to the health and welfare of mammals. Although mammals eventually acquire immunity to nematodes, this breaks down around parturition, which renders periparturient mammals susceptible to re-infection and an infection source for their offspring. Nutrient supplementation reduces the extent of periparturient parasitism, but the underlying mechanisms remain unclear. Here, we use a genome wide approach to assess the effects of protein supplementation on gene expression in the small intestine of periparturient rats following nematode re-infection. METHODOLOGY/PRINCIPAL FINDINGS: The use of a rat whole genome expression microarray (Affymetrix Gene 1.0ST) showed significant differential regulation of 91 genes in the small intestine of lactating rats, re-infected with Nippostrongylus brasiliensis compared to controls; affected functions included immune cell trafficking, cell-mediated responses and antigen presentation. Genes with a previously described role in immune response to nematodes, such as mast cell proteases, and intelectin, and others newly associated with nematode expulsion, such as anterior gradient homolog 2 were identified. Protein supplementation resulted in significant differential regulation of 64 genes; affected functions included protein synthesis, cellular function and maintenance. It increased cell metabolism, evident from the high number of non-coding RNA and the increased synthesis of ribosomal proteins. It regulated immune responses, through T-cell activation and proliferation. The up-regulation of transcription factor forkhead box P1 in unsupplemented, parasitised hosts may be indicative of a delayed immune response in these animals. CONCLUSIONS/SIGNIFICANCE: This study provides the first evidence for nutritional regulation of genes related to immunity to nematodes at the site of parasitism, during expulsion. Additionally it reveals genes induced following secondary parasite challenge in lactating mammals, not previously associated with parasite expulsion. This work is a first step towards defining disease predisposition, identifying markers for nutritional imbalance and developing sustainable measures for parasite control in domestic mammals.

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.

Immunization of cattle with a combination of purified intimin-531, EspA and Tir significantly reduces shedding of Escherichia coli O157:H7 following oral challenge.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen that can cause gastrointestinal disease with potentially fatal consequences as a result of systemic Shiga toxin activity. Cattle are the main reservoir host of EHEC O157 and interventions need to be developed that prevent cattle colonization or limit shedding of the organism from this host. EHEC O157 predominately colonizes the bovine terminal rectum and requires a type III secretion system (T3SS) for adherence and persistence at this site. A vaccine based on concentrated bacterial supernatant that contains T3S proteins has shown some efficacy. Here we have demonstrated that vaccination with a combination of antigens associated with T3S-mediated adherence; the translocon filament protein, EspA, the extracellular region of the outer membrane adhesin, intimin, and the translocated intimin receptor (Tir) significantly reduced shedding of EHEC O157 from experimentally infected animals. Furthermore, this protection may be augmented by addition of H7 flagellin to the vaccine preparation that has been previously demonstrated to be partially protective in cattle. Protection correlates with systemic and mucosal antibody responses to the defined antigens and validates the targeting of these colonization factors.

IgA and IgG antibody responses following systemic immunization of cattle with native H7 flagellin differ in epitope recognition and capacity to neutralise TLR5 signalling.

Systemic immunization of cattle with H7 flagellin results in induction of both H7-specific IgA and IgG antibodies but only partially protects against subsequent colonization with Escherichia coli O157:H7. Recent studies indicate that anti-flagellin antibodies directed against TLR5 binding domains located in the conserved N- and C-terminal domains of flagellin can neutralise TLR5 activation and impair vaccine efficacy. In the current study we determined whether systemic immunization of cattle with H7 flagellin induces antibodies capable of interfering with flagellin-mediated TLR5 activation. Both anti-H7 IgG1 and IgG2 but not IgA antibodies recognised epitopes within the conserved N- and C-terminal domains of H7 flagellin, and purified H7-specific IgG but not IgA was capable of inhibiting H7-mediated TLR5 activation in vitro. These results suggest that (i) IgA and IgG isotypes originated from different populations of B cells and (ii) systemically induced H7-specific IgG but not IgA may impair innate immune responses to E. coli O157:H7 via neutralisation of TLR5 activation and subsequently reduce vaccine efficacy.