Download Biochemical and physiological studies to identify

DEPARTMENT for ENVIRONMENT, FOOD and RURAL AFFAIRS
Research and Development
CSG 15
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 [email protected]
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
Project start date
£ £384,341
01/07/00
Project end date
30/06/03
Executive summary (maximum 2 sides A4)
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 closelyrelated 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.
CSG 15 (Rev. 6/02)
1
Project
title
Biochemical and physiological studies to identify potential
targets for the control of Psoroptes ovis
DEFRA
project code
OD0536
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.
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. A 329bp fragment of DNA, amplified from P.
cuniculi genomic DNA, was shown to have a high homology with human cytosol aminopeptidase. The activity
and gene was characterised, and shown to be a typical cytosolic leucine aminopeptidase in the M17 group of
metalloproteinases. It was not possible to produce sufficient recombinant protein in bacterial expression systems,
but it may be possible to express the sequences in different eukaryotic and prokaryotic systems. 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.
CSG 15 (Rev. 6/02)
2
Project
title
Biochemical and physiological studies to identify potential
targets for the control of Psoroptes ovis
DEFRA
project code
OD0536
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)
3
Project
title
Biochemical and physiological studies to identify potential
targets for the control of Psoroptes ovis
DEFRA
project code
OD0536
Scientific report (maximum 20 sides A4)
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. -Glutamyl transpeptidase is involved in the degradation of glutathione, and
that an important role in cysteine metabolism.
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
OD0536
Objective 3: Role of mite enzymes and bacteria in extra-oral digestion and pathology defined.
Lipase activity was detected in the soluble fraction of P. ovis; but whether this activity was of bacterial or mite
originis unclear. The cDNA library was screened using nested PCR for a lipase gene, using primers designed from
Drosophila melanogaster, but no lipase gene could be detected in this material.
The cDNA libraries were screened for the lysozyme gene, using degenerate primers designed from the silkworm
Bombyx mori and the tick lysozyme sequence. Lysozyme is important in antibacterial activity and is a typical
enzyme from insect salivary glands.
On the skin surface, four different areas were removed from five P. ovis infected sheep. These four areas were the
initial site of infection, a mid-lesion sample, a sample from the leading edge of the lesion, and an area of skin
unaffected by mites. The samples were assayed for changes in lipase, lysosome (Figure 3), aminopeptidase
(Figure 4), aspartic peptidase (Figure 5) and esterase, and the amount of protein was determined. Each piece of
skin was measured, weighed and the different physical characteristics recorded. A general observation was that
total enzyme activity was lower in the areas of skin that had not been infected with mites.
The analysis of these data sets is being completed as part of a PhD thesis. The relative activities across each lesion
will be further assessed, and a profile of the enzyme ‘ecology’ across a P. ovis infected lesion established. Further
work is required to determine if the enzymes present are specifically of mite origin rather than part of the
pathology response or produced by bacteria associated with the lesion. Many enzymes of possible gut origin are
also allergens (see review article), and one major allergen from Dermatophagoides pteronyssinus (Der P 1), is a
cysteine proteinase common to other mite species (Dermatophagoides farinae, Lepidoglyphus destructor,
Dermatophagoides microceras, and Euroglyphus maynei).
Objective 4: Investigate the role played by lipase producing bacteria in the mite midgut on the
digestive physiology of the parasite.
A part of this objective was to characterise bacterial populations from mites in field outbreaks of disease, but due
to logistical problems, and the intervention of FMD, this prove difficult. Efforts were therefore focused on trying
to identify the role these bacteria may play in mite pathogenicity. Lesion material and associated mites were
sampled and the relationship between the bacteria on the surface of the skin and the P. ovis mite investigated. Each
lesion was arbitrarily cut in to 3 pieces, and each area was designated 1) initial site of infection, 2) mid-lesion and
3) leading edge. P. ovis associated bacterial and sheep skin 16s DNA was amplified by PCR using universal
bacterial 16s primers. The PCR products were cloned into a TOPO TA vector and screened by PCR amplification
using M13 primers. Novel sequences were detected by RFLP using HAE III and MSP I restriction enzymes
(Figure 6) and the corresponding plasmids were sequenced. 16s sequences were matched to their closest
neighbours in existing databases using BLAST and RDP software. The most closely related species to the ones
identified are listed in Table 1. From this work, a novel RFLP profiling by identifying differences of one or more
bands after digestion with both restriction enzymes has been developed. The bacteria flora present on the skin of
uninfested sheep includes Shphingomonas mali, Afipia genosp. and Alpha proteobacterium.
To date, nine different species of bacteria have been identified as being associated with the P. ovis mite and the
corresponding skin scrape. 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 (see below). As far as can be ascertained, gramnegative lipase producing bacteria do not appear to be associated with the mites.
CSG 15 (1/00)
5
Project
title
Biochemical and physiological studies to identify potential
targets for the control of Psoroptes ovis
DEFRA
project code
OD0536
Burkholderia (previously known as Pseudomonas) cepacia is a gram-negative bacteria that is resistant to many
antibiotics, and is able to metabolise many substrates. It is generall y found in soil and other moist environments. It
has emerged as an important opportunistic pathogen to humans affected by cystic fibrosis and
immunocomprimised patients and interestingly causes ovine mastitis. B. cepacia has an unusual metabolism and is
able to degrade chlorinated aromatic substances for use as a carbon source. B. cepacia is able to prevent leaf and
stem blight caused by the fungus Alternaria, by inhibiting spore germination.
A few Corynebacterium species are part of the natural flora of humans, but they are occasionally isolated as
opportunistic pathogens in patients who are immunocomprimised. Corynebacterium confusum has been isolated
from two patients with foot infections and from a blood culture of a third patient. Corynebacterium capitovis sp. is
a gram-positive bacterium and was isolated from skin scrapings from an infected head of a sheep. Whether any of
these bacteria species is able to cause the pathology associated with sheep scab is still to be ascertained.
Objective 5: Establishment of optimal nutritional requirement of the sheep scab mite
The data from this project have been used to characterise the optimal nutrient requirements for P. ovis. The enzyme
complement described will enable modelling of its potential effect on both the sheep-derived molecules and
bacteria associated with the lesion. 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, and these data will
be used to enhance the potential for in vitro mite culture.
Objective 6: Identification and development of techniques for improving the in vitro culture of
the sheep scab mite.
Mite feeders were designed based upon previous preliminary studies (Figure 7). The feeder keeps 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 the skin surface dwelling bacteria. Mites for the trials were
washed prior to being placed in the chamber. Each chamber contained 20 mites of mixed sex and age and each trial
was repeated 5 times. Analysis of the results was performed using Kaplan-Meier survival analysis to test the
probability of a mite surviving for a given time after the start of the experiment. The following nutritional
supplements were tested.






Live and dead E. coli and M. luteus, Serratia marcessens,
Burkholderia sp. mixed with and without lipase,
Foetal calf serum,
Blood mixed with live bacteria and lipase,
Yeast
An artificial blood meal containing 20% protein.
The 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 the nutritional supplement of the artificial blood meal that was 20% protein based (Figures 8
and 9).
Objective 7: Evaluation of selected potential targets for immune, chemical and biological control
strategies.
The central approach from the parallel project (OD0537) has been the gradual refinement of mite sub-cellular
fractions as immunogens. Assays for enrichment of enzyme activities in the protective fractions have been
conducted. Aminopeptidase and aspartic peptidase activities were present in mite fractions (Figure 9), and
enriched in some but could not be correlated with a protective effect in sheep.
CSG 15 (1/00)
6
Project
title
Biochemical and physiological studies to identify potential
targets for the control of Psoroptes ovis
DEFRA
project code
OD0536
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.
Four veterinary/medical disinfectants (Virkon S, Hibiscrub, Dettol and Fresh-shield) were assessed regarding their
efficacy to reduce bacterial numbers on the skin of live sheep and assess the duration of any bactericidal activity.
This study was carried out prior to future studies involving mite challenge assessing the relationship between sheep
skin bacteria and the pathogenesis of Psoroptes ovis. Each disinfectant was diluted according to the manufacturers
instructions and massaged into the withers of two sheep (where the fleece had been clipped to 2.0 cm), ensuring
that disinfection occurred at least 5.0cm into the unclipped fleece at the periphery of the area. Two sheep remained
as untreated controls. Skin washings (2.0ml sterile phosphate buffered saline) were collected from two sites within
the disinfected area from all sheep on Days 0, +1,+2, +3 and +4 post disinfection, diluted 1/10 and 1/100 in sterile
PBS and immediately cultured (including the undiluted washing) onto 10% SBA and incubated aerobically at 37C
for 24hrs. After this time the total number of colonies was counted for each dilution together with differential
counts for all colony types present. Representatives of each colony were also gram stained and deep frozen in NA
for future identification. The numbers of colony forming units (CFUs), assumed to be the result of a single
bacterial cell were counted for each dilution of skin washing. Numbers of CFUs were multiplied by the dilution
factor and the mean of the numbers of CFUs for three dilutions calculated. In order to compare the results between
disinfectants, individual sheep and skin washing sites numbers of CFUs were scored (Table 2). Results are shown
in Table 3. The bacterial flora of sheepskin comprised 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. Yet others presented relatively high populations with a relatively
greater number of species. If the skin bacterial flora is essential for the pathogenesis of scab such variation in
bacterial numbers may reflect an individual‘s susceptibility to scab. Under the conditions of this study the
disinfectants assessed failed to render sheep skin totally sterile. The above preliminary study was undertaken at the
request of an Ethics committee and in accordance with Home Office regulations as a prelude to any scab mite
infection studies. Due to the outcome of the preliminary results from this trial and possible animal welfare
implications from proposed mite-disinfectant studies, a further possilbe follow up study was not allowed to
proceed for welfare reasons.
Publications/Presentations arising from project
Billingsley, P. F. Digestion in the sheep scab mite, Psoroptes ovis and targets for immune control. Invited seminar
to the Medical and Veterinary Special Interest Group of the Royal Entomological Society. 2001.
Hamilton, K. A., Nisbet, A. J., Lehane, M. J., Taylor, M.A., Billingsley, P. F. A physiological and biochemical
model for digestion in the ectoparasitic mite, Psoroptes ovis (Acari, Psoroptidae). International Journal for
Parasitology 33:773-785.
Hamilton, K.A., A.J. Nisbet, M.J.Lehane, M.A.Taylor, P.F. Billingsley. A model for digestion in the sheep scab
mite P. ovis. Royal Entomological Society University of Aberdeen September 9th – 12th 2001.
Hamilton, K.A., A.J. Nisbet, M.J. Lehane, M.A. Taylor, P.F. Billingsley. A model for digestion in the sheep scab
mite P. ovis. Scottish Universities Molecular Parasitology meeting, Kindrogan15-17th May 2001.
Hamilton, K.A., A.J. Nisbet, M.J. Lehane, M.A. Taylor, P.F. Billingsley. A model for digestion in the sheep scab
mite P. ovis. British Society for Parasitology meeting Manchester UMIST 7-9th April 2003.
Nisbet, A. J., Billingsley P. F. 2002. Characterisation of aminopeptidase activity in scab mites, Psoroptes spp.
Insect Biochemistry and Molecular Biology 32:1123-1131
CSG 15 (1/00)
7
Project
title
Biochemical and physiological studies to identify potential
targets for the control of Psoroptes ovis
Figure 1.
Sequence of the mite M17 aminopeptidase gene and inferred protein.
Primer sequences are highlighted in yellow, zinc-binding and active
sites in purple.
GGGAAGCAGTGGTATCAACGCAGTGTGGCCATTATGGCCGGGGAAATTTAATTCATTTATTAGTCAAAATT
CATTTGAATATAACCAACACACATTGACATTTTTTACCAATAATCGTCAATTAAATACATCATCAATTATA
1 ATG AAT AAA AAT AAA GCC ACA CTT ATC GGT GTA TTT GAG AAT AGT ACG AAT
1 M
N
K
N
K
A
T
L
I
G
V
F
E
N
S
T
N
61 TTC ATC TTT ACA CCG ACT GGT GAG AAA ATC AAT TCT TCA ATC GGT GGT GTC
21 F
I
F
T
P
T
G
E
K
I
N
S
S
I
G
G
V
121 CAA TTA AAT ATC GTT GGT CCA GTG AAA AAA TGT AAA GTT CGA ATA TTA TAT
41 Q
L
N
I
V
G
P
V
K
K
C
K
V
R
I
L
Y
181 CCA GAA TAT CCA ATT GTA GGT GTT GTT GGT CTT GGT CCT GAT AAT GCA ACA
61 P
E
Y
P
I
V
G
V
V
G
L
G
P
D
N
A
T
241 CTG GAA GAA TTG GAT GAA AAA TCG GAA AAT ATT CGT TCA GCC GTT GCT ACC
81 L
E
E
L
D
E
K
S
E
N
I
R
S
A
V
A
T
301 GCA TTA CGT GAT CTT GGA TCA ATT GAA GAA ATC AAT GTT GAT GGA TGC TTG
101 A
L
R
D
L
G
S
I
E
E
I
N
V
D
G
C
L
361 GCA GCA TCT GAA GGT GCT AAT CTT GGT TTA TAT TAT TTT GAT GAA TTG AAA
121 A
A
S
E
G
A
N
L
G
L
Y
Y
F
D
E
L
K
421 CTC AAA AAG AAT TTG GTT AAA GTT AAT TTG TTA TCA AAC GAA GAA TCA GAT
141 L
K
K
N
L
V
K
V
N
L
L
S
N
E
E
S
D
481 TGG AAT GCC GGC GTT GTA TTG TCA AAT GGA CAA AAT TTT TGC CGT ACA CTG
161 W
N
A
G
V
V
L
S
N
G
Q
N
F
C
R
T
L
541 CCG GCT AAT TTA ATG ACT CCA ACT AAA TTT GCT GAA ATC GCC AGT GCC ACT
181 P
A
N
L
M
T
P
T
K
F
A
E
I
A
S
A
T
601 TTG GAT GTC ACG GTA AAT GTT CGT GAT AAA GCA TGG GCT GAA TCA ATG AAA
201 L
D
V
T
V
N
V
R
D
K
A
W
A
E
S
M
K
661 TTT TTG AGT GTT GCT AAA GGT TCA GAT GAA CCA CCA GTT TTT CTC GAA ATT
221 F
L
S
V
A
K
G
S
D
E
P
P
V
F
L
E
I
721 AAT GCA CCT GAC ACA AAA CCA TTG GTG TTT GTT GGC AAA GGA ATA ACA TTT
241 N
A
P
D
T
K
P
L
V
F
V
G
K
G
I
T
F
781 GGA ATT TCA TTG AAA CCA TCA TCC AAT ATG GAT AAA ATG CGT GCC GAT ATG
261 G
I
S
L
K
P
S
S
N
M
D
K
M
R
A
D
M
841 GCT AAT GTT GTC AGT ACA ATT TAT ACG TTG GCC ACA AAA AAA TCT CCA GTC
281 A
N
V
V
S
T
I
Y
T
L
A
T
K
K
S
P
V
901 GGA TTG ATA CCG TTG TGT GAA AAT TTG CCA AGC GGA AAA GCC AAT AAA CCT
301 G
L
I
P
L
C
E
N
L
P
S
G
K
A
N
K
P
961 GTC ACT GCA ATG AAT GGA AAA ACT ATT CAA GTT GAT AAC ACT GAT GCT GAA
321 V
T
A
M
N
G
K
T
I
Q
V
D
N
T
D
A
E
1021 ATT TTG GCC GAT GCT CTC TGT TAC GCA CAT CAA TTT AAC CCA TTT TTA ATT
341 I
L
A
D
A
L
C
Y
A
H
Q
F
N
P
F
L
I
1081 GCC ACA TTG ACA GGT GCT ATT AAT GTT GCG CTA GGC TCA GCC GCT ACC GGT
361 A
T
L
T
G
A
I
N
V
A
L
G
S
A
A
T
G
1141 ACT ACG AGC AAA TAT TGG ACT ATG TTG CAA AAA TGT GGT GTA GAA ACT GGT
381 T
T
S
K
Y
W
T
M
L
Q
K
C
G
V
E
T
G
1201 TGG CGT ATG CCT TTG TTT AAT CAT TAC ACT AAA CAG ACC ACT GAT AGC CAA
401 W
R
M
P
L
F
N
H
Y
T
K
Q
T
T
D
S
Q
1261 CTC TGT AAT ATT GGT AAA TAT GCA GGG CAA GGT GGA AGC TGC ATA GCA GCC
421 L
C
N
I
G
K
Y
A
G
Q
G
G
S
C
I
A
A
1321 CGC GAA TTC GTC ACC TGC AAT AAT TGG ATC CAT TTT GAT ATT GCT GGT GTG
441 R
E
F
V
T
C
N
N
W
I
H
F
D
I
A
G
V
1381 AAA ACT GAA ATA GTT TAT CTC TCC AAA GGA ATG GCT GGC CGA CCA TTA CGC
461 K
T
E
I
V
Y
L
S
K
G
M
A
G
R
P
L
R
1441 AAA TTC GTT GAA GAG ATT TTC GAA AAC AAA GCC TTC TAA GCA ATA AAT TTA
481 K
F
V
E
E
I
F
E
N
K
A
F
*
1501 CAT TAT CAA ACG ATA ATT TTA GAG AAG ATG AAT TGA AAT AAA AAA TTT TAT
1561 AAA AAA AAA AAA AA
CSG 15 (1/00)
DEFRA
project code
8
AAA
K
ATT
I
GAT
D
TAT
Y
GGT
G
GAC
D
GCG
A
GAA
E
ATG
M
TTG
L
ATG
M
CAT
H
GAT
D
GGT
G
AAT
N
GGT
G
GGG
G
ATG
M
GTA
V
GAT
D
TTG
L
GCT
A
ATG
M
ACA
T
TTT
GAT
D
GAA
E
GTA
V
AAT
N
GTA
V
CCT
P
CCA
P
AGC
S
GAA
E
TCC
S
GGT
G
TAT
Y
AGC
S
GGT
G
ATC
I
GAT
D
CGT
R
GAC
D
TTT
F
CGC
R
GCT
A
TTC
F
GAA
E
TTG
L
ATT
TCT
S
AAA
K
AGT
S
GAA
E
CGA
R
AAA
K
GCA
A
CTT
L
AAT
N
AAG
K
TCA
S
AAT
N
GGT
G
GCT
A
ATA
I
GTT
V
CTA
L
ATC
I
TGC
C
ATG
M
GAT
D
CTC
L
AAC
N
GTG
V
TCA
TGA CTT AAA
OD0536
Project
title
Biochemical and physiological studies to identify potential
targets for the control of Psoroptes ovis
DEFRA
project code
OD0536
Figure 2. Expression of mite M17 aminopeptidase in E. coli. Top – coomassie-stained
SDS-PAGE gel showing induced protein expression with a band at the expected
position (~55-60 kDa; red arrows). Bottom – Western blot showing the 55-60 kDa band
(red arrow) detected using an anti-His antibody.
Induced
Uninduced
MW (kDa)
75
50
30
15
Induce
d
Uninduced
MW
(kDa)
50
30
15
CSG 15 (1/00)
9
Project
title
Biochemical and physiological studies to identify potential
targets for the control of Psoroptes ovis
Figure 3:
DEFRA
project code
OD0536
Lysozyme activity in skin and lesion samples from three sheep (a-c). Samples
1-5 are described above. Lysozyme activity in skin of sheep not infected with
mites was below the level of the detection. 1 Enzyme Unit is defined as the
activity that will produce at A450nm of 0.001 per min at pH 6.24 @25C using
Mirococcus luteus as a substrate.
EU of lysozyme per mg of protein
400
350
300
250
A
B
200
C
1 50
1 00
50
0
1
2
3
4
5
Sam ple num ber
Figure 4:
Aminopeptidase activity in skin and lesion samples from three sheep (a-c).
Samples 1-5 are described above. Activity is plotted as a percentage of sample 1
(uninfected area). 2.5mM LpNA was used as substrate. Sheep 1 had no piece of
skin that acted as a control.
% of control skin activity
2500
2000
1500
A
B
1000
C
500
0
site1
1
site2
site1
2
site2
site1
3
site2
Sam ple num ber
CSG 15 (1/00)
10
site1
4
site2
site1
5
site2
Project
title
Biochemical and physiological studies to identify potential
targets for the control of Psoroptes ovis
Figure 5:
DEFRA
project code
Aspartic peptidase activity in skin and lesion samples from three sheep (a-c).
Samples 1-5 are described above. H-Pro-Thr-Glu-Phe-Phe(NO2)-Arg-Leu-OH was
used as substrate.
0.08
0.07
0.06
Change in OD
0.05
0.04
A
B
0.03
C
D
0.02
0.01
0
-0.01
1
2
3
-0.02
Sheep number and site
CSG 15 (1/00)
OD0536
11
4
Project
title
Figure 6.
Biochemical and physiological studies to identify potential
targets for the control of Psoroptes ovis
DEFRA
project code
OD0536
Genotyping and species identification of bacteria associated with the lesion and with
Psoroptes ovis using the restriction enzymes HAE III and MSP I.
Gel 1 displays 4 novel
sequences. Samples C and D display identical banding patterns. However A, B and C show
different banding patterns. Gel 2 indicates only 2 novel sequences, F-H present identical
banding patterns compared to I.
MW (bp)
CSG 15 (1/00)
A
B
C
GEL 1
D E
MW
MW
12
F
G
GEL 2
H
I
MW (bp)
Project
title
Biochemical and physiological studies to identify potential
targets for the control of Psoroptes ovis
DEFRA
project code
Table 1. Bacterial flora present on the skin and lesion surfaces of two sheep infected
with Psoroptes ovis*.
Site 1
Common to skin & mite
Burkholderia sp.
Mite
Staphylococcus aureus
uncultured rape
rhizosphere
Skin
Acinetobacter
Site 2
Burkholderia sp
Bradyrhizobium sp
Sphingomonas sp.
Site 3
Burkholderia sp.
Beta proteobacterium
Site 4
Site 5
Not applicable
Not applicable
Nesterenkonia sp
Escherichia coli
Shigella flexnari
Not applicable
Not applicable
Nesterenkonia sp
Acinetobacter sp.
Jeotgalibacillushalotolerans
Staphylococcus aureus sp.
Paracoccus sp.
Staphylococcus aureus
Site 1
Common to skin & mite
None
Site 2
Staphylococcus sp
Site 3
Nesterenkonia sp
Psychrobacter sp.
Site 4
Not applicable
Mite
Escherichia coli
Pseudomonas syringae
Escherichia coli
Burkholderia sp.
Shigella sp.
Staphylococcus aureus
Psychrobacter sp.
Staphylococcus sp.
Corynebacterium
confusum
Not applicable
Site 5
Not applicable
Not applicable
*
Pseudomonas sp.
Pseudomonas sp.
Skin
Corynebacterium confusum
Psychrobacter glacincola
Acinetobacter
Staphyloccous vitulus
Nesterenkonia sp
Pesudomonas sp.
Psychrobacter sp.
Staphylococcus aureus
Pesudomonas sp.
Psychrobacter sp.
Acinetobacter sp.
The bacterial flora was also examined from the skin of a sheep not infected with P. ovis. The
species identified in this control included Shphingomonas mali, Afipia genosp and Alpha
proteobacterium.
CSG 15 (1/00)
13
OD0536
Project
title
Biochemical and physiological studies to identify potential
targets for the control of Psoroptes ovis
Figure 7. Plexi-Glass Feeding Device for in vitro culture of mites
CSG 15 (1/00)
14
DEFRA
project code
OD0536
Project
title
Biochemical and physiological studies to identify potential
targets for the control of Psoroptes ovis
DEFRA
project code
Figure 8. Survival curves of Psoroptes ovis fed on an artificial blood meal (blue) and supplied
with no nutritional supplement (orange). Results show a significant prolongation
of survival when mites are provided with the artificial blood meal (Chi sq= 12.5, df
=1, p= 0.000396).
1.0
0.8
entage alive
0.6
0.4
0.2
0.0
0
2
4
6
8
Time (days)
CSG 15 (1/00)
15
OD0536
Biochemical and physiological studies to identify potential
targets for the control of Psoroptes ovis
Figure 9.
Survival curves of Psoroptes ovis fed on a diet containing Burkholderia spp.
bacteria (orange) supplied with no nutritional supplement (blue). Results show a
significant decline in survival when mites are provided with Burkholderia spp.
bacteria (Chi sq= 11.2, df =1, p= p=0.00083).
0.6
0.8
1.0
Project
title
0.0
0.2
0.4
Percentage alive
0
CSG 15 (1/00)
2
4
Time
(days)
16
6
8
DEFRA
project code
OD0536
Project
title
Biochemical and physiological studies to identify potential
targets for the control of Psoroptes ovis
DEFRA
project code
Table 2: Colony Forming Units (CFU) Scoring System
Score
0
+
++
+++
++++
+++++
CFU Range
0
1-10
11-100
101-1000
1001-10,000
10,001-100,000
Table 3 : Colony Forming Units CFU Scores for Four Disinfectants with Time.
Disinfectant
Sample
Days after Treatment
0
1
2
3
4
Control
5321 /A
5321/B
5449/A
5449/B
++
++
++++
++++
++++
+
+++++
+++++
+
++
++++
+++++
+++++
++
++++
+++
+++
+
+++++
+++++
Virkon
5368/A
5368/B
5373/A
5373/B
+++
++++
+++
+++
+
0
++
++
+
+
++++
+++
+++
++
+++
+
+
++
++++
++++
Hibiscrub
5571 /A
5571/B
5360/A
5360/B
+
++++
++
++
++++
++
0
++++
++
+++
0
0
+++
++
++
0
++
+++
0
0
Dettol
5313/A
5313/B
5394/A
5394/B
++++
+++
+
0
+++
+++++
++
+++
+++
+++++
+++
++
+++++
+++++
+++
++++
++++
++++
++++
+++
FreshShield
5355/A
+++++
+
+
+
++
5355/B
5387/A
5387/B
++++
++++
++
+
+++
++
+
++++
+++
+++
++
++
++
+++
+++
CSG 15 (1/00)
17
OD0536