Dermal immune responses against Psoroptes ovis in two cattle breeds and effects of anti-inflammatory dexamethasone treatment on the development of psoroptic mange.

Psoroptic mange is a common disease of livestock, caused by Psoroptes ovis. Compared to Holstein-Friesian (HF) cattle, the Belgian Blue (BB) cattle breed is highly susceptible to the infestation. However, the mechanism for this difference is still unclear. To determine the factors responsible for this breed susceptibility, the immune response to P. ovis was studied in experimentally infested BB and HF cattle, using clinical signs, histology, immunohistochemical profiling and gene expression analysis of skin biopsies. The mite numbers and lesion area of BB cattle were greater than in HF during the whole study period. Significant influxes of eosinophils in the epidermis and dermis were detected in comparison with the pre-infestation samples in both breeds, with significantly higher eosinophils in BB at 6 weeks post infestation (wpi). Mast cell numbers were unaffected at all stages of infestation in HF, but were significantly elevated relative to pre-infestation in BB cattle at 2 and 6 wpi. The more pronounced cutaneous eosinophilia and higher IL-4 levels at 6 wpi in BB cattle suggest that a Th2-type immune response is underlying the higher susceptibility of the BB breed. In naturally infested BB cattle, development of the psoroptic mange lesions and eosinophils and CD3+ T cell areas were severely depressed after anti-inflammatory treatment with dexamethasone. Together, these results suggest that a stronger Th2-type immune response to P. ovis causes the skin lesions in psoroptic mange in BB cattle and that local anti-inflammatory treatment could potentially be an alternative to control the pathology caused by this parasite.

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.

Transcriptome-based analysis of putative allergens of Chorioptes texanus.

BACKGROUND: Mites of the genus Chorioptes are non-burrowing and cause mange in a wide range of domestic and wild animals including cattle, horses, sheep, goats, panda, moose, camelids, mydaus and alpacas. Molecular biology and host-parasite interactions of Chorioptes texanus are poorly understood, and only a few C. texanus genes and transcript sequences are available in public databases including the allergen genes. METHODS: Chorioptes texanus RNA was isolated from mites, and the transcriptome of C. texanus was analyzed using bioinformatics tools. Chorioptes texanus unigenes were compared with the allergen protein sequences from the mite allergen database website to predict the potential allergens. Chorioptes texanus putative allergen unigenes were compared with hydrolase genes by building a C. texanus hydrolase gene library with the best match of the homologous sequences. Three allergen genes were cloned and expressed, their recombinant proteins were purified and their allergenic activities were preliminarily investigated. RESULTS: Transcriptome sequencing (RNA-Seq) of C. texanus was analyzed and results demonstrated that 33,138 unigenes were assembled with an average length of 751 bp. A total of 15,130 unigenes were annotated and 5598 unigenes were enriched in 262 KEGG signaling pathways. We obtained 209 putative allergen genes and 34 putative allergen-hydrolase genes. Three recombinant allergen proteins were observed to induce different degrees of allergic reactions on rabbit skin. CONCLUSIONS: The present transcriptome data provide a useful basis for understanding the host-parasite interaction and molecular biology of the C. texanus mite. The allergenic activities of recombinant Euroglyphus maynei 1-like (Eur m 1-like) protein, Dermatophagoides ptreronyssinus 1-like (Der p 1-like) protein and Dermatophagoides ptreronyssinus 7-like (Der p 7-like) protein were preliminarily investigated by intradermal skin test. Meanwhile, differences in eosinophil counts were observed in different injected sites of the skin. The identification of putative allergen genes and hydrolase genes offers opportunities for the development of new diagnostic, prevention and treatment methods.

Der f 35: An MD-2-like house dust mite allergen that cross-reacts with Der f 2 and Pso o 2.

BACKGROUND: Dermatophagoides farinae is a source of airborne house dust mite (HDM) allergens. We elucidated IgE-reactive allergens from D. farinae by two-dimensional immunoblotting-based allergenome analysis, and identified one new allergen, named Der f 35, that possesses IgE-binding capacity comparable to that of Der f 2. The aim of this study was to clarify the allergenic capacity of new HDM allergen Der f 35. METHODS: We cloned der f 35 from D. farinae mRNA and produced recombinant Der f 35 in Escherichia coli. The IgE-binding capacity of Der f 35 and its cross-reactivity with group 2 allergens from D. farinae and Psoroptes ovis were determined by enzyme-linked immunosorbent assay (ELISA) and ELISA inhibition assays, respectively. RESULTS: The deduced amino acid sequence for der f 35, which possesses the MD-2-related lipid-recognition domain, showed higher identity with group 2 allergens from P. ovis (61.5%) and Blomia tropicalis (50.7%) than with Der f 2 (40.8%). Der f 35 showed IgE-binding frequencies of 77.5% (31/40) for the native form upon allergenome analysis and 51.4% (18/35) for recombinant structure by ELISA. Der f 35 showed cross-reactivity with Der f 2 and Pso o 2 in reaction with HDM-allergic patients' IgE by ELISA inhibition assay. CONCLUSION: Der f 35 is a candidate major allergen from D. farinae, which is more similar to group 2 allergens from sheep scab mite and storage mites. Der f 35 could be responsible for the cross-reactivity among group 2 mite allergens.

Evaluating troponin C from Psoroptes cuniculi as a diagnostic antigen for a dot-ELISA assay to diagnose mite infestations in rabbits.

The mite Psoroptes cuniculi is globally widespread and has a serious impact on commercial rabbit breeding. In China, diagnosis of P. cuniculi is currently based on conventional clinical methods that entail numerous disadvantages, including their failure to diagnose subclinical infections. Hence, alternative measures are required, and dot-ELISA is one of the most promising strategies. We cloned and expressed the recombinant P. cuniculi troponin C gene for use as a basis for novel dot-ELISA assay to detect P. cuniculi infections in rabbits. This amplified sequence encoded a 153 amino acid protein of 17·6 kDa and theoretical pI 4·18 without signal peptide. The recombinant troponin C of P. cuniculi is an outer membrane protein and may also be a new P. cuniculi allergen. Results of dot-ELISA test showed that this novel assay had more than 90% sensitivity but low specificity in distinguishing infections with P. cuniculi or Sarcoptic scabiei, despite very high agreement between observers (97-99%; κ values ranged from 0·95 to 0·98 for inter- and intra-observer variability test). This study showed that this novel method, at present, lacks diagnostic utility. Therefore, although simple serological assays such as dot-ELISA show great promise as diagnostic tools, we suggest that troponin C is not a suitable diagnostic antigen candidate.

Efficacy of the d-phenothrin/pyriproxyfen association against mites in naturally co-infested rabbits / Eficácia da associação de d-fenotrina e piriproxifen no controle de ácaros em coelhos naturalmente co-infestados

The aim of the present study was to evaluate the efficacy of the d-phenothrin/pyriproxyfen association against Psoroptes ovis, Cheyletiella parasitivorax, and Leporacarus gibbus infestations in naturally co-infested rabbits. Twenty crossbreed (New Zealand White x California) rabbits concurrently infested by the three mite species were randomly divided in two groups. All rabbits presented with hyperemia, erythema and formation of crusts in the ear canals caused by P. ovis. Infestations by both C. parasitivorax and L. gibbus were considered asymptomatic in all animals.Ten animals were treated with a 4.4% d-phenothrin and 0.148% pyriproxyfen spray formulation until have their body surface uniformly sprayed, including external ear canals. The other ten rabbits remained untreated, serving as control group. Observations were done on days +7, +14, +21, +28, and +35 post-treatment. The d-phenothrin/pyriproxyfen association showed 100% efficacy against the three mite species and was responsible for the remission of psoroptic mange lesions on treated animals. No signs of intoxication were observed. The results indicate that d-phenothrin/pyriproxyfen spray formulation in a single application is an effective and clinically safe option for the control of different mite infestations in rabbits.

O objetivo do presente estudo foi avaliar a eficácia da associação de d-fenotrina e piriproxifen no controle de infestações simultâneas por Psoroptes ovis, Cheyletiella parasitivorax e Leporacarus gibbus em coelhos naturalmente co-infestados. Vinte coelhos mestiços (Nova Zelândia Branco x Califórnia) infestados simultaneamente pelas três espécies de ácaros foram divididos aleatoriamente em dois grupos. Todos os coelhos infestados por apresentavam eritema, hiperemia e formação de crostas nas orelhas, causados por P. ovis. Infestações simultâneas por C. parasitivorax e L. gibbus foram considerados assintomáticas em todos os animais. Dez animais foram tratados com uma formulação spray contendo d-fenotrina a 4,4% e piriproxifen a 0,148%, pulverizando toda a superfície corporal de forma uniforme, incluindo a face interna das orelhas. Os outros 10 coelhos não foram t ratados, sendo mantidos como grupo controle. Os animais foram avaliados nos dias 7, 14, 21, 28 e 35 pós-tratamento. A associação de d-fenotrina e piriproxifen foi 100% eficaz no controle das três espécies de ácaros e foi responsável pela remissão das lesões de sarna psoróptica nos animais tratados. Não foram observados sinais de intoxicação. Os resultados indicam que a formulação spray de d-fenotrina e piriproxifen em uma única aplicação é uma opção clinicamente segura e eficaz no controle de infestações por ácaros em coelhos.

Vaccination with recombinant actin from scab mites and evaluation of its protective efficacy against Psoroptes cuniculi infection.

The mite Psoroptes cuniculi is globally widespread and has a serious impact on commercial rabbit breeding. Current treatment methods are based on chemotherapy. Because of the disadvantages of these methods, alternative measures are required, and vaccination is one of the most promising strategies. Here, we cloned and expressed the recombinant P. cuniculi actin gene (rPc-act). Antiserum levels against rPc-act in rabbits were used to locate actin distribution in mite sections. Challenge trials were carried out to evaluate the immunity protection of rPc-act in rabbits, with antibody levels determined by ELISA. Sequence analysis of this gene fragment showed 89·26% and 84·91% identity to Sarcoptes scabiei and Mayetiola destructor sequences, respectively. Immunohistochemistry showed rPc-act to locate widely throughout the mites, especially in feet and muscle tissues. Recombinant P. cuniculi actin with QuliA adjuvant was used to immunize six rabbits. Each animal was challenge-infested with 25-50 adult mites. Although IgE levels showed no significant difference to controls, IgG levels were significantly higher, and clinical development showed no significantly different severity of lesions in vaccinated rabbits than in the controls. This study showed that rPc-act is a muscular isotype actin and has no clinical protective efficacy against P. cuniculi.

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.

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.

Infestation of sheep with Psoroptes ovis, the sheep scab mite, results in recruitment of Foxp3(+) T cells into the dermis.

Regulatory T cells (Tregs) play a central role in maintenance of immune homeostasis by controlling harmful immune responses to inappropriate antigens and are thought to play a key role in modulating hypersensitivity reactions. Infestation of sheep with Psoroptes ovis results in a pronounced cutaneous hypersensitivity-type response, which appears to be crucial for mite survival. We hypothesize that (i) Tregs are involved in sheep scab lesions and (ii) Treg responses may crucially affect lesion development and subsequent mite survival. Foxp3 is a key transcription factor required for generation and maintenance of Tregs in rodents and humans, and is the most widely used marker for Tregs in these species. In this study, we sequence ovine foxp3 and show that it exhibits a high degree of homology with foxp3 from other species. Using a validated immunohistochemical staining technique, we demonstrate that infestation of sheep with P. ovis results in an influx of Foxp3(+) T cells into the skin. Future work will investigate the regulatory function of ovine Foxp3(+) T cells and determine whether the quality of the Treg response to P. ovis plays a role in individual susceptibility to the mite.

Gene expression profiling of ovine keratinocytes stimulated with Psoroptes ovis mite antigen--a preliminary study.

Sheep scab is caused by the noninvasive mite, Psoroptes ovis, which initiates a profound pro-inflammatory skin response leading to lesion development. To investigate these early events between the skin and the parasite, primary ovine epidermal keratinocyte cultures were generated and challenged with mite derived antigens. The kinetics of the mRNA response of these cells were monitored by microarray. The results indicated that the cells responded within 1 h of challenge, with a significant increase in the pro-inflammatory cytokine IL-8. This result was confirmed by real-time RT-PCR, and showed that IL-8 up-regulation was maximal at 1 h but declined to pre-stimulation levels at 24 and 48 h. The IL-8 mRNA response to mite wash antigens containing secretory and/or excretory proteins was also investigated and compared to the response to whole mite antigen. These studies revealed that the mite wash antigen, at a challenge dose of 10 microg/mL, was markedly more potent and induced significantly higher levels of IL-8 mRNA than the same concentration of whole mite antigen. These results are discussed in relation to mite establishment and survival on the ovine host.

Molecular characterization, expression and localization of a peroxiredoxin from the sheep scab mite, Psoroptes ovis.

The sheep scab mite, Psoroptes ovis, induces an intensely pruritic exudative dermatitis which is responsible for restlessness, loss of appetite and weight loss. Within the first 24 h of infection, there is a rapid inflammatory influx of eosinophils and apoptosis of the keratinocytes at the site of infection. The former cell type is capable of a sustained respiratory burst, toxic products of which may directly damage the mite and also contribute to lesion formation. Analysis of a P. ovis expressed sequence tag (EST) database identified a number of antioxidant enzyme-encoding sequences, including peroxiredoxin (thioredoxin peroxidase EC 1.11.1.15), all of which may help the mite endure the potentially toxic skin environment. A full length sequence encoding Po-TPx, a protein of 206 amino acids which showed high homology to a peroxiredoxin from the salivary gland of the tick Ixodes scapularis, was amplified from P. ovis cDNA. Recombinant Po-TPx was expressed in bacteria and antiserum to this protein was used to localize native Po-TPx in mite sections. Peroxiredoxin was localized, amongst other sites, to a subpharyngeal region in mite sections. The recombinant protein was recognized by sera from sheep infested with the mite suggesting that it may be secreted or excreted by the mite and interact with the host immune response.

Psoroptes ovis: identification of vaccine candidates by immunoscreening.

Serum from successful vaccine trials against the sheep scab mite, Psoroptes ovis, was used to immunoscreen a cDNA library constructed from mixed-stage and gender P. ovis to identify potential recombinant vaccine candidates. Immunodominant recombinant proteins recognised by IgG in these sera were selected for further analysis. Two candidates were identified in this way; a catchin-like protein (CLP) and a novel mu class glutathione S-transferase (GST). Both candidates were expressed in bacteria as recombinant proteins, the GST as an active enzyme, and combined with four other recombinant allergens in a multi-component recombinant vaccine. Strong serum IgG responses were induced in sheep against each of the components of the recombinant vaccine, however, the protective efficacy of the vaccine could not be determined because of variability in the establishment of a challenge infection.

Stimulation of the in vitro migration of ovine eosinophils by factors derived from the sheep scab mite, Psoroptes ovis.

The ectoparasitic astigmatid mite Psoroptes ovis causes sheep scab, a highly contagious, severe allergic dermatitis associated with damage to the fleece and hide, loss of condition and occasional mortality. The scab lesion is characterized by a massive infiltration of eosinophils that begins very rapidly after infection. This paper reports the finding that mite-derived factors directly enhance the migration of ovine eosinophils in vitro. Significant (p < 0.01) and dose-dependent (r = 0.972 +/- 0.018 (SD)) activity was initially identified in whole mite extracts, by comparison with medium controls in an assay based on modified Boyden chambers and ovine bone marrow target cells. Similar pro-migratory activity (p < 0.005; r = 0.928 +/- 0.069 (SD)) was detected in washes containing mite excretory/secretory material. By direct comparison with migration ratios (n = 3) for defined chemotactic (rmeotaxin = 3.430 +/- 0.360 (SD)) and chemokinetic (rminterleukin-5 = 0.982 +/- 0.112 (SD)) stimuli it was determined that the activity in both mite extracts (0.992 +/- 0.038 (SD)) and mite washes (0.969 +/- 0.071 (SD)) was chemokinetic. Subsequent experiments (n = 3) in which live mites were incorporated directly into the in vitro assay system indicated that they produced factors that significantly (p < 0.001) enhanced eosinophil migration to a degree directly related to mite numbers (r = 0.993 +/- 0.005 (SD)). The identity of the factor(s) responsible is uncertain, but their presence suggests that mites may be capable of directly activating eosinophils in vivo, and raises the possibility that mites could directly influence, perhaps even initiate, the rapid early tissue eosinophilic response observed in experimental sheep scab infections.

Immunocytochemistry of Psoroptes cuniculi stained by sera from naive and infested rabbits: preliminary results.

Immunocytochemistry was used to identify possible target antigens in the digestive system of Psoroptes cuniculi. Sera from three recently acutely infested rabbits, from rabbits with a mild long lasting infestation, and from a rabbit with repeated mite infestations and no longer able to maintain a population of P. cuniculi were used to determine any antibody specificity to the mite digestive system. The reactivity of these sera was compared with sera from three un-infested animals. The different pool of sera targeted different mite antigens; in particular, sera from the resistant rabbit and the chronically infested rabbits reacted with gut cells, faecal material and cuticle, while sera from the recently infested rabbits reacted with gut contents, faecal material and cuticle of the parasites but not with gut cells. Finally, sera from un-infested rabbits did not demonstrate any specificity to P. cuniculi antigens reacting only with mite gut contents in a weak manner. These preliminary data suggest the presence of antibodies induced in the host blood by infection, which act against the parasite by binding to antigen at the surface of its gut.

Eukaryotic expression of recombinant Pso o 1, an allergen from Psoroptes ovis, and its localization in the mite.

A cDNA encoding the immunogen Pso o 1 from Psoroptes ovis was obtained by polymerase chain reaction (PCR) amplification. The amplicon contained the entire coding sequence for the prepro-enzyme in an open reading frame (ORF) of 966 bp. This gene encoded a predicted protein of 322 amino acids (aa) with 64% aa identity (80% similarity) to the major house dust mite faecal allergen Der f 1. The pro-enzyme form of Pso o 1 was expressed as a recombinant protein in the Pichia pastoris-eukaryotic expression system. Maturation of the recombinant pro-enzyme by autocatalytic activation was not observed, and such maturation could not be achieved using a number of techniques known to activate recombinant Der p 1 and Der f 1 expressed in the same system. Serum raised against recombinant Pso o 1 cross-reacted with mature Der p 1 and allowed Pso o 1 to be immunolocalized to the gut of P. ovis.

Molecular characterization, expression and localization of tropomyosin and paramyosin immunodominant allergens from sheep scab mites (Psoroptes ovis).

cDNAs encoding the immunodominant allergens tropomyosin and paramyosin were amplified from RNA extracted from the sheep scab mite Psoroptes ovis. The tropomyosin cDNA contained an open reading frame (ORF) of 852 bp which encoded a predicted protein with 98% and 97% identity to the house dust mite allergens Der f 10 and Der p 10 respectively. The complete paramyosin ORF generated by RT-PCR was 2625 bp in length and encoded an 875aa predicted protein of 102.6 kDa with 97%, 95% and 89% identity to the paramyosins of Dermatophagoides pteronyssinus (Der p 11), Sarcoptes scabiei and Blomia tropicalis (Blo t 11) respectively. Full length tropomyosin and truncated and full-length paramyosin were expressed as recombinant proteins. IgG and IgE in sera from sheep with a 6-week duration primary infestation of P. ovis did not detect either full-length or truncated recombinant paramyosin. IgG in both infested and naïve sheep sera detected recombinant tropomyosin, suggesting cross-reactivity to tropomyosin and to other invertebrate species to which the sheep may have been exposed. Staining with antibodies directed against tropomyosin and paramyosin was observed throughout sections of P. ovis. Staining was especially prevalent in the anterior sections of the mites, possibly associated with locomotory muscles in this region.

Immune responses to Staphylococcus aureus and Psoroptes ovis in sheep infected with P. ovis--the sheep scab mite.

In sheep, lesions caused by Psoroptes ovis, the sheep scab mite, may become colonized by Staphylococcus aureus. The present study compares clinical signs, lesional area and the immune response to P. ovis and S. aureus in P. ovis-infested sheep with and without secondary S. aureus infection. No differences were detected in the clinical signs or lesional areas in the S. aureus-positive and -negative sheep. However, 6 weeks after infestation an IgG but not IgE isotype antibody response to S. aureus was detected in the S. aureus-positive but not the S. aureus-negative group of sheep. This response targeted S. aureus antigens with molecular weights of approximately 36, 38, 50 and 65 kDa. In addition, 6 weeks after infestation an IgE response to P. ovis was detected in the S. aureus-positive but not the S. aureus-negative group of sheep.