Research and Development s6

Project
title / Biochemical and physiological studies to identify potential targets for the control of Psoroptes ovis
/ DEFRA
project code / ODO 536

Department for Environment, Food and Rural Affairs CSG 15

Research and Development

Final Project Report

(Not to be used for LINK projects)

Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to
Project title / Biochemical and physiological studies to identify potential targets for the control of Psoroptes ovis
DEFRA project code / OD0536
Contractor organisation and location / Veterinary Laboratories Agency, New Haw, Addlestone, Surrey, KT15 3NB
Total DEFRA project costs / £ £384,341
Project start date / 01/07/00 / Project end date / 30/06/03
Executive summary (maximum 2 sides A4)
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CSG 15 (Rev. 6/02) 4

Project
title / Biochemical and physiological studies to identify potential targets for the control of Psoroptes ovis
/ DEFRA
project code / ODO 536

Sheep scab is a highly contagious parasitic disease of sheep caused by the mite Psoroptes ovis. Infection causes intense itching and wool loss with high morbidity and occasional mortality in severely affected animals. The disease has considerable welfare implications. Control has traditionally involved the application of chemical pesticides by plunge dipping, although injectable endectocides are now in widespread use. There exists considerable public concern over the toxicity and safety of sheep dips and chemical control methods generally. There is therefore an urgent need to investigate alternative methods of sheep scab control.

Whilst the pathophysiology of the sheep scab mite and methods of chemotherapy have been well researched, there is little information on the basic physiology and biochemistry of P. ovis. The size of the mite has usually precluded physiological studies on mite organ systems, as conducted on insects. However, digestive enzymes from P. ovis have been described at the biochemical and gene levels, and assays established for routine detection of activities. Furthermore, until recently, there was a similar paucity of information on mite feeding habits. Lack of such fundamental data has prohibited the development of specific targeting drugs, research into vaccine-based control, and biological alternatives to chemicals.

An important handicap to the generation and application of basic physiological data on P. ovis is the inability to grow the organism in vitro. Thus, studies so far, have had to use mites grown in vivo on infected sheep or closely-related species, such as P. cuniculi from rabbits.

This project was designed to investigate the digestive physiology and nutritional biology of the mite with the aim of targeting inhibitors of mite digestion. The project also set out to investigate the role of digestive enzymes in modifying the mite’s immediate environment and thereby causing pathology (n common with medically important mites such as house dust mite, scabies mites), and the role of bacteria, already identified as part of the gut microflora of the mite, to ascertain their role in nutrition and digestion. It was obvious that such studies would require, to some extent, the development of an in vitro culture system for P. ovis. The obligatory parasitic existence of the mite made this a key and challenging objective.

One of the major outputs of the project was a review article on the digestive physiology of the sheep scab mite, Psoroptes ovis, and other, related astigmatic mites entitled ‘A physiological and Biochemical Model for Digestion in the Ectoparasitic Mite, Psoroptes ovis (Acari: Psoroptidae) published in the International Journal for Parasitology. The review covered the structure of mite digestion systems and the various digestive enzyme types characterised from mite extracts. It also attempts to relate these enzymes to diet utilisation, allergenicity of mites, and the relationship between symbiotic micro-organisms, pathogenic bacteria and digestion in arthropods and relevance to mite digestive physiology.

A γ-Glutamyl transpeptidase in P. ovis was also identified, but attempts to clone the gene and obtain usable sequence data were not convincing.

Previous work has identified lipase-producing, or lipophilic bacteria, in the gut microflora of the sheep scab mite but it was unclear what role they played in mite nutrition and digestion. Lipase activity was detected in the soluble fraction of P. ovis, but despite screening the cDNA library using nested PCR, no lipase gene could be detected in this material. It remains unclear whether the lipase enzymes present are specifically of mite origin, part of the pathology response, or produced by bacteria associated with the lesion.

The bacteria flora present on the skin of uninfested sheep includes Shphingomonas mali, Afipia genosp., and Alpha proteobacterium. Examination of lesion material and mites revealed nine different bacterial species identified as being associated with the P. ovis mite and the corresponding skin area. These are Acinetobacter spp., Burkholderia spp. Beta Proteobacterium, Bradyrhizobium spp., Escherichia coli, Corynebacterium confusum, Psychrobacter sp., Pseudomonas sp., Nesterenkonia sp, Shigella flexnari, Jeotgalibacillus halotolerans, and Staphylococcus aureus. In all cases, one pathogenic bacterium species appears always to be associated with the mite and the corresponding scab material. Burkholderia sp. and Corynebacterium confusum are both pathogenic to humans. Whether the mites are releasing these bacteria onto the surface of the skin and initiating an immune response is still unclear. It is however apparent that the mites are not utilising the bacteria as a food source. It is thought therefore, that gram-negative lipase producing bacteria do not appear to be associated with the mites.

Data from this project have been used to characterise the optimal nutrient requirements for P. ovis. The model described in the review article strongly suggests that the optimal nutritional state is achieved by the mite causing massive modification of the skin environment, such knowledge is important in enhancing the potential for in vitro mite culture.

In vitro culture studies were conducted in mite feeders that kept the mites in contact with the test substrate whilst allowing ventilation and observation. The temperature was maintained at 33°C, equivalent to the fleece and skin surface temperature. A variety of media were tested, mainly bacterial, in order to establish whether the mites were grazing on skin-surface dwelling bacteria. Results indicated that the mites do not use bacteria as a food source. There was a significant decline in the survival rates of the mites when the nutritional supplement was Burkholderia sp. However there was a significant rise in survival with nutritional supplementation with artificial blood meal that was 20% protein based.

Assays for enrichment of enzyme activities within protective mite sub-cellular fractions from the sheep immunology project, OD0537, were conducted. Whilst aminopeptidase and aspartic peptidase activities were present in mite fractions and enriched in some, these could not be correlated with a protective effect in sheep.

A role for bacteria in the scab lesion pathology has been previously advocated, and as such, a trial testing antibacterial compounds for their role in lesion development was completed. The trial was conducted to determine if disinfectants could reduce bacterial numbers on the skin of live sheep and assess the duration of any bactericidal activity. It was shown that the bacterial flora of ovine sheep skin comprised of a number of species with the diversity of species and numbers of bacteria varying between individual sheep and sampling sites. Some sheep presented relatively low bacterial numbers of poor species diversity. Under the conditions of this study the disinfectants assessed failed to render sheep skin totally sterile. For ethical reasons, further research involving use of disinfectants on mite-infested sheep was not pursued.

An understanding of the physiology of mite digestion is an essential component of the research into potential and alternative methods of control. The results of this project provide a model for the digestion in P. ovis and the potential for targeted therapies.

The mite digestion model can be summarised as follows. The mite when first established on the host probably feeds on the loose stratum corneum and on lipid secretions present. As the mites wander across the surface of the skin, allergenic and enzymatically active material is deposited on the skin causing an inflammatory response. Damage to the skin results in release of serum exudates and bacterial infection and the mites feed on the ensuing nutrient “soup” present. The food is ingested and digested in the midgut by a process of pinocytosis into type II digestive cells where proteolysis is initiated by lysosomally derived endopeptidases (aspartyl and cysteine proteinases, possibly metalloproteinases) and is followed by lysosomal and and cytosolic exopeptidases (cysteine proteinases and aminopeptidases). Proteolysis may also involve luminal enzymes derived from secretory Type I cells and membrane bound enzymes. Within the entire digestive system of the mite are large populations of luminal bacteria, which may play a dietary role yet to be determined.

CSG 15 (Rev. 6/02) 4

Project
title / Biochemical and physiological studies to identify potential targets for the control of Psoroptes ovis
/ DEFRA
project code / ODO 536
Scientific report (maximum 20 sides A4)
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Press the DOWN arrow once to move to the next question.

CSG 15 (Rev. 6/02) 4

Project
title / Biochemical and physiological studies to identify potential targets for the control of Psoroptes ovis
/ DEFRA
project code / ODO 536

Objective 1: Review existing knowledge on mite digestive physiology

A thorough review of the literature concerning the digestive physiology of the sheep scab mite, Psoroptes ovis, and other, related astigmatic mites was conducted leading to the publication of a review article entitled ‘A physiological and Biochemical Model for Digestion in the Ectoparasitic Mite, Psoroptes ovis (Acari: Psoroptidae) by Hamilton, K. A., Nisbet, A. J., Lehane, M. J., Taylor, M.A., Billingsley, P. F. in the International Journal for Parasitology (see reference list below). In the review, the structure of mite digestion systems is reviewed, with cross-references to tick physiology for comparison, with the aim of identifying specific cell types within the gut wall of the mite. Various digestive enzyme types characterised from mite extracts (and where relevant, insects) e.g. Der p 1 (a cysteine proteinase), and substrates, digestive inhibitors, sub-cellular sites, physiological roles have all been reviewed. Where possible, these enzymes have been related to diet utilisation and allergenicity of mites. The role of peptidase activities in modulating their allergenicity was of particular relevance to the research work conducted. The relationship between symbiotic micro-organisms, pathogenic bacteria and digestion in arthropods was also reviewed and its relevance to mite digestive physiology examined. (114 references cited).

Objective 2: Characterisation of the biochemical and enzymatic activity of mite digestion.

The digestive processes in Psoroptes spp are believed to involve intracellular digestion by lysosomal endopeptidases (cathepsin-like aspartic and cysteine proteinases) followed by further processing by intra and/or extracellular exopeptidases including aminopeptidases. Soluble and membrane-bound aminopeptidase activities have been demonstrated in extracts of P. cuniculi and P.ovis. Aminopeptidase activity was eluted as a single peak (85-116kDa) from soluble extracts of P. cuniculi by gel filtration FPLC. Native electrophoresis of the concentrated eluates from FPLC demonstrated a single band of aminopeptidase activity. Degenerate, oligonucleotide primers were designed using conserved areas of amino acid sequence and a 329bp fragment of DNA was amplified from P. cuniculi genomic DNA. Analysis of this fragment revealed a nucleotide sequence coding for a protein sequence with high homology (63% amino acid identity) with human cytosol aminopeptidase. The activity and gene has now been characterised, and is a typical cytosolic leucine aminopeptidase in the M17 group of metalloproteinases. It has a preference for leucine and methionine substrates, is inhibited by leucinethiol, bestatin, Arphamenine A and 1,10-phenanthroline, Zn2+, Cu2+ Ni2+, Co2+ and activated by Mn2+ and Mg2+. Activity was detected as a single major band on native gels; and as a single peak in size exclusion chromatography of 85-116kDa. Using primers to conserved regions around the active and zinc-binding sites, the molecular sequence of the same gene (Fig. 1) has been characterised, and in preparation for possible vaccination trials, the mite LAP sequence in E. coli has been expressed (Figure 2). Producing sufficient recombinant protein in the absence of bacterial protein background has so far proven impossible, but attempts will be made to express the sequences in different eukaryotic and prokaryotic systems.

Both soluble and membrane-bound aminopeptidase activities were present in P. cuniculi and part of this activity was attributable to a M1 leucine aminopeptidase. For this enzyme, clones were available containing full length P. ovis M1 LAP amplified from the P. ovis cDNA library, but the 3' end contained an inverted poly-T sequence. This was thought to be a result of codon bias in the library, causing differential amplification during the PCR steps of the library construction. Degenerate primers to conserved motifs of rat, Drosophila, C. elegans, and Aedes amino acid sequences were used to amplify a P. ovis M1 LAP fragment, but attempts to gain full-length sequence from the P. ovis cDNA library have proven unsuccessful.

A γ-Glutamyl transpeptidase in P. ovis has also been identified, but attempts to clone the gene and obtain usable sequence data were not convincing. g-Glutamyl transpeptidase is involved in the degradation of glutathione, and that an important role in cysteine metabolism.

Objective 3: Role of mite enzymes and bacteria in extra-oral digestion and pathology defined.