WO2001011365A1 - Method for detection of mite infection - Google Patents
Method for detection of mite infection Download PDFInfo
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- WO2001011365A1 WO2001011365A1 PCT/AU2000/000930 AU0000930W WO0111365A1 WO 2001011365 A1 WO2001011365 A1 WO 2001011365A1 AU 0000930 W AU0000930 W AU 0000930W WO 0111365 A1 WO0111365 A1 WO 0111365A1
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- antigen
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- 230000037390 scarring Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 206010040882 skin lesion Diseases 0.000 description 1
- 231100000444 skin lesion Toxicity 0.000 description 1
- 238000010181 skin prick test Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 108010036927 trypsin-like serine protease Proteins 0.000 description 1
- 208000027930 type IV hypersensitivity disease Diseases 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
- G01N2333/43552—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects
- G01N2333/43582—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from insects from mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/976—Trypsin; Chymotrypsin
Definitions
- the present invention relates to methods or diagnostic assays for the detection of mite infections in animals and humans, in particular, the detection of the scabies mite, Sarcoptes scabiei, as well as relating to diagnostic test kits for carrying out those assays and to agents used in those assays, including diagnostic reagents, antibodies and antigens.
- Substitute sheet Australia have shown that the number of mites present in infected pigs is negatively correlated with the degree of rubbing and the extent of skin lesions at slaughter (Cargill et al, 1997) . Therefore the examination of skin scrapings for live mites is not a reliable test for pigs with significant hypersensitivity reactions since these animals often have only low numbers of mites and no encrusted lesions to scrape.
- Substitute sheet main disadvantages are the non-specificity of the response and often labile nature of the antigens used.
- ELISA Enzyme-linked immunosorbent assay
- 2 c peptide including the function to bind to an antibody to the specified/particular protein, also fall within this definition.
- Modifications can include, but are not limited to, additions, deletions or substitutions of amino acid residues, substitution with compounds that mimic amino acid structure or function, as well as the addition of chemical moieties such as amino and acetyl groups.
- the modifications can either be deliberate or can be 0 accidental such as through mutation in hosts which produce the specified peptide. All of these modifications are included as long as the peptide retains its biological function, including the function of binding to an antibody to the specified/particular peptide.
- immunodominant epitope as used herein in relation to a protein, means an antigenic determinant of the protein recognised by antibodies of an animal to which such antibodies will mount or help an immune response.
- immunodominant region as used in relation to a protein, means a region of the protein containing an immunodominant epitope.
- the present invention provides a method for the detection of mite infections in animals including humans, in particular, the detection of the scabies mite, Sarcoptes scabiei, in a sample taken from the animal, which method comprises contacting said sample with either:
- Substitute sheet (a) an antibody/antibodies directed to, or that binds with: (i) one or more antigens in contact with the immune system of the animal early in said infection; or (ii) one or more antigens induced in the host animal by said infection (auto-antigen(s)), or (iii) a combination of said antigen(s) and auto-antigen(s), and detecting the binding to said sample of said antibody/antibodies to indicate the presence in said sample of said antigen(s) and/or auto-antigen(s); or
- the antigen(s) is selected from: a mite alpha-amylase, or a peptide with substantially the same amino acid sequence as a mite alpha-amylase, or an immunodominant region or epitope of a mite alpha-amylase or a peptide with substantially the same amino acid sequence thereof; a mite trypsin, or a peptide with substantially the same amino acid sequence as a mite trypsin, or an immunodominant region or epitope of a mite trypsin or a peptide with substantially the same amino acid sequence thereof; a mite trypsin-like protease, or a peptide with substantially the same amino acid sequence as a mite tryps
- the present invention provides a diagnostic test kit suitable for detecting mite infections in animals including humans, in particular, detecting the scabies mite, Sarcoptes scabiei, comprising a device for contactmg and binding a sample taken from the animal, including dipsticks or wells, and a detection means for communicating with the sample, wherein the detection means comprises either:
- the present invention provides a diagnostic reagent for detection of mite infection in an animal, including humans, comprising an antibody/antibodies directed to, or that binds with: (i) one or more antigens in contact with the immune system of the animal early in said infection; or (ii) one or more antigens induced in the host animal by said infection (auto-antigen(s)), or (iii) a combination of said antigen(s) and auto-antigen(s), wherein said antigen(s) and/or auto-antigen(s) is in accordance with the first aspect of the invention, and said antibody/antibodies is/are labelled so as to permit detection of the reaction between said antibody/antibodies with said antigen(s) and/or auto-antigen(s).
- the present invention provides an antibody/antibodies directed to one or more antigens according to the first aspect of the invention, or to one or more antigens induced in the host animal by said infection according to the first aspect of the invention (auto-antigen(s)), or to a combination of said antigen(s) and said auto-antigen(s).
- the method/diagnostic test kit/diagnostic reagent is characterised in that:
- the antibody is a monoclonal antibody
- the antigen is ⁇ -amylase of Sarcoptes scabiei, or ⁇ -amylase of Dermatophagoides Spp., in particular, of Dermatophagoides pteronyssinus or D. farinae, or ⁇ -amylase of Lucilia cuprina or ⁇ -amylase of another arthropod species; or
- the antigen is trypsin of Sarcoptes scabiei, or trypsin of Dermatophagoides Spp., in particular, of Dermatophagoides pteronyssinus or D. farinae, or trypsin of Lucilia cuprina or trypsin of another arthropod species; or
- the antigen is trypsin-like protease of Sarcoptes scabiei, or trypsin-like protease of Dermatophagoides Spp., in particular, of Dermatophagoides pteronyssinus or D. farinae, or trypsin-like protease of Lucilia cuprina or trypsin-like protease of another arthropod species; or
- the antigen is serine protease of Sarcoptes scabiei, or serine protease of Dermatophagoides Spp., in particular, of Dermatophagoides pteronyssinus or D. farinae, or serine protease of Lucilia cuprina or serine protease of another arthropod species; or
- Substitute sheet the antigen is excretory/secretory product (ESP) of Lucilia cuprina or other such arthropod, in particular, larval excretory/secretory product (LESP) of Lucilia cuprina; or
- the detection is by ELISA testing or fluorescent/luminol binding assay or radioimmunoassay, or precipitation or agglutination, more particularly, by an ELISA test specific to Sarcoptes scabiei; or
- the detection is by a skin based hypersensitivity test, more particularly, by a skin test specific to Sarcoptes scabiei; or
- the animal is selected from pigs, horses, cows, sheep, dogs, cats, wombats, buffalo, deer, rabbits, donkeys, goats, foxes, camels and humans.
- the test kit/ diagnostic reagent is characterised in that the antibody/monoclonal antibody is labelled so as to change colour upon reaction with an antigen in the sample taken from the animal.
- the antibodies in particular, the monoclonal antibodies, of the present invention, as well as the diagnostic agents of the present invention, may be prepared as described herein or by known methods (Kohler and Milstein, 1975; Harlow and Lane, 1988).
- the Enzyme Linked Immuno-Sorbent Assay can be adopted to determine the levels of either antigen or antibody in body fluids or tissue extracts at sensitivities well below those of other tests.
- An antibody detection by ELISA may involve the use of an antigen bound to a substrate so as to enable binding of any antibody present in skin washings or serum extracts from putitatively infected animals. The bound antibody would then be detected with an antibody conjugated to a reporter molecule.
- amylase antigens are suitable for an ELISA format test, or for skin testing (skin prick tests are
- Substitute sheet commonly used in allergy testing in pets and humans may be used in the detection of mite infections in a range of hosts.
- the invention enables the detection of the scabies mite, Sarcoptes scabiei in the skin of various species of host mammals.
- the invention relies on the identification of certain mite molecules which are present in the skin during infection and which produce strong antibody responses in the host.
- One such molecule is the enzyme, alpha amylase, which is produced by mites.
- Another molecule is transferrin a normal blood constituent of the host that is apparently changed by the mite to stimulate the production of auto-antibodies which are characteristic of scabies infections.
- the detection assay is based on assays that use antibodies to detect the presence of the mite molecules or of host antibodies against these molecules.
- the immunoassay uses a reporter molecule bound to an antibody or another detection agent and this signals the presence of the mite by a colour change or other detectable event.
- the assay is more sensitive than current diagnostic techniques.
- the invention uses one or more antigens or auto-antibodies, or a combination thereof, in various forms of detection assay including, but not limited to, an enzyme linked immunosorbent assay or other forms of detection assay.
- the assay will detect either, the presence of the antibody/antigen/auto-antibody in the skin or of antibody to the antigen(s) or auto-antibody in the serum of infected animals or man.
- the presence of the antibody/antigen/auto-antibody will be signalled by a colour change or other detectable event above a threshold level in an antigen or antibody detection assay. Animals giving a signal above this level will be deemed to be infected with Sarcoptes scabiei.
- Substitute sheet antigens was carried out using an anti Pig-HRP conjugate and chromogenic detection using DAB.
- the major proteins were serum albumin, immunoglobulin G (heavy and light chain), transferrin, haptoglobin and hemoglobin alpha-chain (see Figure 1).
- serum albumin serum albumin
- immunoglobulin G heavy and light chain
- transferrin transferrin
- haptoglobin hemoglobin alpha-chain
- Dilute antigen (transferrin; bovine source; SIGMA) in Carbonate coating buffer to a concentration of lO ⁇ g/ml and coat each well on a microlitre plate with 50 ⁇ l, allow protein to bind overnight at 4°C.
- Transferrin is the major serum glycoprotein that transports iron to most tissues in mammals (Steverding, et al. 1995). Thus, the host iron binding molecules are apparently important to mite metabolism.
- the published sequence for house dust mite alpha-amylase is K YX ⁇ PHF IGX RSN IT XLM E. Due to the low amount of alpha-amylase available from a Sarcoptes source, an alternate bacterial source of alpha-amylase was trialed. Purified alpha-amylase was obtained commercially (bacterial source, Bacillus species type II-A; SIGMA) and tested for reactivity to pigs by ELISA. In testing of a total of 44 pigs there was a difference in the reactivity of the two groups of pigs to alpha-amylase. The mean of the mange negative pigs + 2 S.D. gave an antibody cut off titre of 300. We found that the protein alpha-amylase was able to distinquish 100% of mange positive pigs from negative controls (Figure 4).
- Substitute sheet In order to obtain larger quantities of an ⁇ -amylase related to the mite enzyme, an excretory/secretory preparation from the blowfly, Lucilia cuprina (Sandeman et al., 1990) was tested for activity. Enzyme activity was confirmed using a commercial substrate (Amylase 3, Sigma Diagnostics 577-3) and by reactivity with sera from mange infected pigs. The blowfly larval ⁇ -amylase was then isolated by extraction on Q Sepaharose (Pharmacia) and the amylase active fractions were pooled for ELISA testing. The ELISA was carried out as outlined above with the exception of two monoclonal antibodies substituting for the anti-pig IgG conjugate.
- mice were mouse anti-porcine IgGl (Serotec, MCA635) and rat anti-mouse IgGl-HRP conjugate (MCA336P) and both were used at a 1 :2000 dilution.
- Pig serum samples used in the ELISA were obtained from abattoir slaughtered pigs diagnosed as having mange or other diseases (acute and chronic pneumonia, pleurisy, ileitis and pleuropneumonia) post mortem ( Figure 5).
- D. pteronyssinus mites were obtained commercially from CSL, Melbourne, Australia, while D. farinae mites were sourced from Allergon AB, Angelholm, Sweden. Extracts were prepared by placing 1 gram of mites in a 50ml plastic tube with 20ml of cold PBS. The tube was vortexed until a homogenous solution of mites and buffer was achieved, the tube was then placed on ice for 6 hours with periodic vortexing every 40 minutes. The homogenate was centrifuged for 10 minutes at 30,000g. The supernatant was aliquoted into 1ml volumes and stored at -70 °C.
- L. cuprina Lucilia cuprina larval excretion/secretion product
- the larvae of L. cuprina were obtained from a laboratory colony maintained in the Parasite Control Laboratory, Department of Agriculture, La Trobe University.
- the flies were originally obtained from a natural infection of fine wool Merino sheep in southern Victoria, Australia.
- the colony was supplemented annually with wild-type flies from natural infections to prevent in-bred mutations.
- Crude LESP was prepared using a method described by Sandeman et al. (1990).
- LESP was freeze dried, desalted to distilled water on a Sephadex G25 column (Pharmacia Biotech), freeze dried again and stored at -70°C. After reconstitution in DDW, two protease inhibitors (EDTA and PMSF at a final concentration of 1 mM) were added to prevent protein degradation.
- the sample (10ml) was loaded onto the column in buffer A and protein elution was carried out using a linear gradient of buffer A to buffer B (0.02M Tris-HCl buffer containing 1M NaCl, pH 8.0).
- the elution gradient was 0-60% Buffer B over 40 minutes at a flow rate of 1ml per minute, then 60-100%) Buffer B over the next 20 minutes at the same flow rate.
- the elution profile was recorded by absorbance at 280nm.
- Alpha-amylase activity was determined using an amylase reagent (AMYLASE 3; Sigma Chemical Company).
- Table 1 Amino acid sequence of 60 and 33 kDa benzamidine sepharose purified proteins from LESP and homologous proteins identified from a BLAST search of the SwissProt. database.
- Substitute sheet Comparing the 60 kDa sequence to the SwissProt. database revealed the highest homology (71%) to two proteins. The first was Trypsin I from crayfish (Astacus astacus) (SwissProt accession number P00765, Titani, et al, 1983) and the second was Collagenolytic Protease 36 kDa precursor from crab (Paralithodes camtschatica) (SwissProt accession number P20734, Klimova, et al, 1990). Trypsin I is one of the five forms of the enzyme known to be present in crayfish. This protein is a serine protease that is more acidic than mammalian trypsin. Collagenolytic protease is a serine protease capable of degrading the native triple helix of collagen.
- Benzamidine Sepharose 6B (Pharmacia Biotech) which specifically binds trypsin and trypsin-like serine proteases, was prepared according to manufacturer's instructions.
- binding buffer 50mM Tris-HCl pH 8.0.
- Substitute sheet Freeze dried LESP was resuspended in 30ml distilled water and desalted on a G-25 column. The desalted LESP fraction was then run down the benzamidine column and the column was washed thoroughly in binding buffer. The bound proteins were eluted using 0.1M glycine-HCl pH 3.0. The bound fraction was immediately neutralised using 1M Tris pH 9.0. The eluted fraction was desalted and concentrated using a pressure-driven, disposable, ultrafiltration device for processing aqueous biological solutions (Ultrafree- PF; Millipore). After reconstitution in DDW, two protease inhibitors (EDTA and PMSF at a final concentration of 1 mM) were added to prevent protein degradation.
- EDTA and PMSF two protease inhibitors
- a blood sample was collected prior to weaning (Day -2).
- pigs in the treated groups were treated with either Ivermectin 1% (w/v) (IVOMEC S/C injection for swine, Merial) or Dectomax (Doramectin IM injection, Pfizer) at the rate of 1 ml/33 kg (300 mcg/kg).
- Pigs in the untreated group remained unmedicated.
- the antibody titres for the three groups of pigs is shown in Table 3 and Figure 8. All three groups showed similar low antibody titres at Day -2 with an average titre of 23. The antibody titres of all three groups at Day -2 were lower than that of the mange free herd comparison (C), which had a mean titre ⁇ S.D. of 72 ⁇ 26. At Day 122 the highest antibody titres were seen for the untreated group (N) with a mean titre ⁇ S.D. of 600 ⁇ 432. In comparison, the Ivermectin group [T(I)] was 225 ⁇ 87 and the Dectomax group [T(D)] was 320 ⁇ 103. It is important to note that the pigs tested at Day -2 were all weaners, and it is reported that pigs of this age do not produce high levels of antibodies to Sarcoptes.
- FIG. 1 SDS-PAGE and staining of the porcine skin scraping protein extract.
- mw Molecular weight standards
- FIG. 3 SDS-PAGE and immunoblotting of a crude Dermatophagoides farinae extract.
- the protein profile of the extract is shown in lanes A and B. Immunoblotting of the dust mite extract was carried out with positive pig sera for Sarcoptes infestation (D) and also with mange negative sera (E). A conjugate control where no primary antibody was tested is shown in lane F. Molecular weight markers (mw) are indicated in Kilodaltons (kDa).
- Substitute sheet Figure 4 Reactivity of pig sera to alpha-amylase (bacterial source) as tested by ELISA. Two groups of pigs being either mange negative or positive were differentiated using a cut-off value of the mean titre of the mange negative pigs plus 2 standard deviations. All positive pigs tested had a value greater than this antibody cut-off titre value of 1/300.
- FIG. 5 Reactivity of abattoir pig sera to insect alpha-amylase as tested by ELISA.
- An alpha-amylase fraction was isolated from a Lucilia cuprina larval excretion/secretion product.
- Sera was obtained from pigs belonging to commercial piggeries that were diagnosed post mortem. Sera was classified into the following disease catagories; disease free, acute pneumonia, chronic pneumonia, pleurisy, and mange positive, The pigs in the pneumonia and pleurisy groups showed no clinical signs of mange post mortem.
- FIG. Gel electrophoresis and immunoblotting of benzamidine purified Lucilia cuprina larval excretory/secretory product (LESP).
- the protein profile of the purified fraction is shown in lane A.
- Benzamidine purified LESP was transferred to PVDF membrane and immunoblotted with positive sera (B, C) and negative sera (D, E). Conjugate controls without primary antibody are shown in lane F.
- Molecular weight markers (mw) are indicated in Kilodaltons (kDa).
- Figure 7 Reactivity of mange negative and positive pig sera to bovine trypsin. Antibody titres were monitored by ELISA.
- Ivermectin for the control of swine scabies relative values of prefarrowing treatment of sows and weaning treatment of pigs.
- Transferrin-binding protein complex is the receptor for transferrin uptake in Trypanosoma brucei. J. Cell Biology
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Abstract
The present invention provides methods or diagnostic assays for the detection of mite infections in animals and humans, in particular, the detection of the scabies mite, Sarcoptes scabiei. The methods involve contacting a sample form the animal with antibodies to: (a) mite alpha-amylase or other antigen in contact with the immune system of the animal early in the infection; and/or (b) transferring or other auto-antigen induced by the infection. Alternatively, the methods involve contacting a sample from the animal with mite alpha-amylase or other antigen in contact with the immune system of the animal early in the infection to indicate the presence in the sample of antibodies to the mite alpha-amylase or said other antigen. The invention also provides diagnostic test kits for carrying out those assays and to agents used in those assays, including diagnostic reagents and antibodies.
Description
METHOD FOR DETECTION OF MITE INFECTION
FIELD OF THE INVENTION
The present invention relates to methods or diagnostic assays for the detection of mite infections in animals and humans, in particular, the detection of the scabies mite, Sarcoptes scabiei, as well as relating to diagnostic test kits for carrying out those assays and to agents used in those assays, including diagnostic reagents, antibodies and antigens.
BACKGROUND OF THE INVENTION
Infection with the mite, Sarcoptes scabiei, which causes mange in pigs, is a major problem in pig health management with estimates of herd infection rates as high as 95% (Cargill et al., 1997, Dobson & Davis, 1992). Mite infections lead to rubbing and damage to fittings, decreased growth rates, decreased fertility and scarring of pig hides (Cargill & Dobson, 1979a; Cargill & Dobson, 1979b; Arends et al., 1990; Martelli & Beghian, 1990). The cost of mange to the producer has been estimated at up to $10/sow/year in herds where the infection is being controlled, and from $20 to $50/sow/year in herds where infection is not controlled (Cutler & Gardiner, 1988). Mange continues to be a significant disease problem and was ranked second behind chronic respiratory diseases in the ten most important diseases affecting the Australian pig industry by the Animal Health Committee Endemic Diseases Working Party (Report to the Australian Health Committee by Endemic Diseases Working Party, 1992). This is despite the presence of highly effective acaricides such as organophosphorous compounds (Cameron, 1984) and Ivermectin (Lee et al., 1980; Primm et al., 1992).
Current diagnosis relies on interpreting variable symptoms such as rubbing, hairloss, visible skin thickening and encrustations and the presence of mites in skin scrapings (Cameron, 1984). In addition, post mortem diagnosis is possible after hair removal at the abattoir when small red papules can be observed on infected pig skins (Pointon et al., 1987; Hollanders & Vercruysse 1990; Davies et al., 1991). The only unequivocal diagnostic is the detection of mites in skin scrapings and this test requires a microscope and a high level of infection so that mites are relatively easily found. Studies in South
Substitute sheet
Australia have shown that the number of mites present in infected pigs is negatively correlated with the degree of rubbing and the extent of skin lesions at slaughter (Cargill et al, 1997) . Therefore the examination of skin scrapings for live mites is not a reliable test for pigs with significant hypersensitivity reactions since these animals often have only low numbers of mites and no encrusted lesions to scrape.
Calculation of the rubbing index (RI) for growing pigs in the herd or an average mange score (AMS) in slaughter pigs have both been shown to be specific and sensitive diagnostic procedures for mite infections above certain threshold levels. Both methods require a degree of training and experience and when infection levels are low they may be confused with other diseases and conditions. At post mortem inspections, for AMS determination papule detection can be confused with insect bites and other rashes and is only a reliable indication of mange above a severity score of about 0.5 (Davies & Moore 1990). Skin tests have been developed which use crude mixtures of mite antigens to test for hypersensitivity reactions in pig skin (Davies & Moon 1990a). Such reactions are sensitive, easy to perform and relatively cheap. They have also been suggested to develop sooner in pigs with low compared to high level infections (Davies & Moon, 1990a). However, a major drawback at present is their lack of specificity with cross reactions due to responses to the antigens of other mite species found in the environment. Mites such as the house dust mite, Dermatophagoides pteronyssinus, which is ubiquitous in the environment and a major cause of human allergy, and Lepidoglyphus destructor, the storage mite, which is associated with stored feedstuffs, could both cause confusing hypersensitivity reactions (Johansson et al, 1991).
Skin reactions to mite antigens have been analysed for a many years in a range of hosts and consequently the timing and types of such reactions are well known. Pigs which are infected with S. scabiei and then skin tested at regular intervals, first show the development of a delayed hypersensitivity reaction then an immediate response (Davies & Moon 1990a). In this study pigs which received low doses of mites developed immediate reactions before those receiving high doses. The exact role of the different types of skin reactions are not known though hypersensitivity reactions certainly cause the major pathological symptoms of the disease rather than the mite itself (Davies & Moon 1990b). The use of skin tests in diagnostics has a number of advantages including on site testing, rapid availability of results and simple protocols which require very little expertise. Their
Substitute sheet
main disadvantages are the non-specificity of the response and often labile nature of the antigens used.
ELISA (Enzyme-linked immunosorbent assay) has been used in the detection of anti- S. scabiei var. suis immunoglobulins in naturally and experimentally infected pigs, using a 5 crude S. scabiei var. suis extract as antigen (Nockler, et al., 1992; Bomstein and Zakrisson, 1993; Bornstein, et al., 1994). Investigators report that the amounts of mite antigen recovered from pig skin scrapings are low. The use of mites isolated from red foxes (S. scabiei var. vulpes) is described as an alternative. The finding that pigs respond with antibody production both during the acute and chronic phases of sarcoptic mange, 10 and that serum antibodies can be detected by serological methods indicates that it is possible to develop sensitive diagnostic tests for the specific diagnosis of this disease (Bornstein and Zakrisson, 1993). The collection of antigen is commonly reported as a major obstacle in the development of a commercial assay.
As used herein, the term "or a peptide with substantially the same sequence as"
15 immediately before a specified/particular peptide, for example, immediately before a reference to mite α-amylase, or the use of the term "or a peptide with substantially the same sequence thereof immediately after a specified/particular peptide, refers to a peptide sequence either identical to, or having functional homology with, the peptide sequence of the specified/particular peptide. It is understood that limited modifications
20. can be made to the specified/particular peptide which result in enhanced function. Likewise, it is understood that limited modifications can be made without destroying the biological function of the specified/particular peptide, including the function of binding to an antibody to the specified/particular peptide. Such minor modification of the sequence which does not destroy the biological function of the specified/particular
2c peptide, including the function to bind to an antibody to the specified/particular protein, also fall within this definition. Modifications can include, but are not limited to, additions, deletions or substitutions of amino acid residues, substitution with compounds that mimic amino acid structure or function, as well as the addition of chemical moieties such as amino and acetyl groups. The modifications can either be deliberate or can be 0 accidental such as through mutation in hosts which produce the specified peptide. All of these modifications are included as long as the peptide retains its biological function, including the function of binding to an antibody to the specified/particular peptide.
Substitute sheet
The term "immunodominant epitope" as used herein in relation to a protein, means an antigenic determinant of the protein recognised by antibodies of an animal to which such antibodies will mount or help an immune response. The term "immunodominant region" as used in relation to a protein, means a region of the protein containing an immunodominant epitope.
The conventional codes for amino acids are used throughout the present specification, ie.:
For Amino Acid Code For Amino Acid Code
Alanine A Leucine L
Arginine R Lysine K
Asparagine N Methionine M
Aspartic Acid D Phenylalanine F
Cysteine C Pro line P
Glutamine Q Serine S
Glutamic Acid E Threonine T
Glycine G Tyrosine Y
Isoleucine I Valine V
With reference to the use of the word(s) "comprise" or "comprises" or "comprising" in the present specification, including the following claims, unless the context requires otherwise, those words are used on the basis and clear understanding that they are to be interpreted inclusively, rather than exclusively, and that each of those words is to be so interpreted in construing the description and/or the claims.
SUMMARY OF THE INVENTION
In a first aspect, the present invention provides a method for the detection of mite infections in animals including humans, in particular, the detection of the scabies mite, Sarcoptes scabiei, in a sample taken from the animal, which method comprises contacting said sample with either:
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(a) an antibody/antibodies directed to, or that binds with: (i) one or more antigens in contact with the immune system of the animal early in said infection; or (ii) one or more antigens induced in the host animal by said infection (auto-antigen(s)), or (iii) a combination of said antigen(s) and auto-antigen(s), and detecting the binding to said sample of said antibody/antibodies to indicate the presence in said sample of said antigen(s) and/or auto-antigen(s); or
(b) one or more antigens in contact with the immune system of the animal early in said infection, and detecting the binding to said sample of said antigen(s) to indicate the presence in said sample of an antibody/antibodies directed to, or that binds with said antigen(s), wherein the antigen(s) is selected from: a mite alpha-amylase, or a peptide with substantially the same amino acid sequence as a mite alpha-amylase, or an immunodominant region or epitope of a mite alpha-amylase or a peptide with substantially the same amino acid sequence thereof; a mite trypsin, or a peptide with substantially the same amino acid sequence as a mite trypsin, or an immunodominant region or epitope of a mite trypsin or a peptide with substantially the same amino acid sequence thereof; a mite trypsin-like protease, or a peptide with substantially the same amino acid sequence as a mite trypsin-like protease, or an immunodominant region or epitope of a mite trypsin- like protease or a peptide with substantially the same amino acid sequence thereof; a mite serine protease, or a peptide with substantially the same amino acid sequence as a mite serine protease, or an immunodominant region or epitope of a mite serine protease or a peptide with substantially the same amino acid sequence thereof; and the excretory/secretory product (ESP) of an arthropod, or a combination thereof; and wherein the auto-antigen(s) is selected from transferrin, haptoglobin or other serine protease homolog or serine-like protease, or a combination thereof.
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In a second aspect, the present invention provides a diagnostic test kit suitable for detecting mite infections in animals including humans, in particular, detecting the scabies mite, Sarcoptes scabiei, comprising a device for contactmg and binding a sample taken from the animal, including dipsticks or wells, and a detection means for communicating with the sample, wherein the detection means comprises either:
(a) an antibody/antibodies directed to, or that binds with: (i) one or more antigens in contact with the immune system of the animal early in said infection; or (ii) one or more antigens induced in the host animal by said infection (auto-antigen(s)), or (iii) a combination of said antigen(s) and auto-antigen(s), wherein said antigen(s) and/or auto-antigen(s) is in accordance with the first aspect of the invention, together with a reagent(s) that is responsive to a reaction between said antibody/antibodies with said antigen(s) and/or auto-antigen(s); or
(b) one or more antigens in contact with the immune system of the animal early in said infection in accordance with the first aspect of the invention, together with a reagent(s) that is responsive to a reaction between said antigen(s) and an antibody/antibodies directed to, or that binds with said antigen(s).
In a third aspect the present invention provides a diagnostic reagent for detection of mite infection in an animal, including humans, comprising an antibody/antibodies directed to, or that binds with: (i) one or more antigens in contact with the immune system of the animal early in said infection; or (ii) one or more antigens induced in the host animal by said infection (auto-antigen(s)), or (iii) a combination of said antigen(s) and auto-antigen(s), wherein said antigen(s) and/or auto-antigen(s) is in accordance with the first aspect of the invention, and said antibody/antibodies is/are labelled so as to permit detection of the reaction between said antibody/antibodies with said antigen(s) and/or auto-antigen(s).
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In a fourth aspect, the present invention provides an antibody/antibodies directed to one or more antigens according to the first aspect of the invention, or to one or more antigens induced in the host animal by said infection according to the first aspect of the invention (auto-antigen(s)), or to a combination of said antigen(s) and said auto-antigen(s).
In preferred embodiments of the first/second/third aspect of the invention, the method/diagnostic test kit/diagnostic reagent is characterised in that:
the antibody is a monoclonal antibody; or
the antigen is α-amylase of Sarcoptes scabiei, or α-amylase of Dermatophagoides Spp., in particular, of Dermatophagoides pteronyssinus or D. farinae, or α-amylase of Lucilia cuprina or α-amylase of another arthropod species; or
the antigen is trypsin of Sarcoptes scabiei, or trypsin of Dermatophagoides Spp., in particular, of Dermatophagoides pteronyssinus or D. farinae, or trypsin of Lucilia cuprina or trypsin of another arthropod species; or
the antigen is trypsin-like protease of Sarcoptes scabiei, or trypsin-like protease of Dermatophagoides Spp., in particular, of Dermatophagoides pteronyssinus or D. farinae, or trypsin-like protease of Lucilia cuprina or trypsin-like protease of another arthropod species; or
the antigen is serine protease of Sarcoptes scabiei, or serine protease of Dermatophagoides Spp., in particular, of Dermatophagoides pteronyssinus or D. farinae, or serine protease of Lucilia cuprina or serine protease of another arthropod species; or
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the antigen is excretory/secretory product (ESP) of Lucilia cuprina or other such arthropod, in particular, larval excretory/secretory product (LESP) of Lucilia cuprina; or
the detection is by ELISA testing or fluorescent/luminol binding assay or radioimmunoassay, or precipitation or agglutination, more particularly, by an ELISA test specific to Sarcoptes scabiei; or
the detection is by a skin based hypersensitivity test, more particularly, by a skin test specific to Sarcoptes scabiei; or
the animal is selected from pigs, horses, cows, sheep, dogs, cats, wombats, buffalo, deer, rabbits, donkeys, goats, foxes, camels and humans.
In preferred embodiments of the second/third aspect of the invention, the test kit/ diagnostic reagent is characterised in that the antibody/monoclonal antibody is labelled so as to change colour upon reaction with an antigen in the sample taken from the animal.
The antibodies, in particular, the monoclonal antibodies, of the present invention, as well as the diagnostic agents of the present invention, may be prepared as described herein or by known methods (Kohler and Milstein, 1975; Harlow and Lane, 1988).
The Enzyme Linked Immuno-Sorbent Assay (ELISA) can be adopted to determine the levels of either antigen or antibody in body fluids or tissue extracts at sensitivities well below those of other tests. An antibody detection by ELISA may involve the use of an antigen bound to a substrate so as to enable binding of any antibody present in skin washings or serum extracts from putitatively infected animals. The bound antibody would then be detected with an antibody conjugated to a reporter molecule.
The potential of a simple, rapid, cheap and reliable assay for scabies mites extends well beyond the pig industry. The largest market would be in the diagnosis of mange in pets followed by the agricultural market and human medical applications. The amylase antigens are suitable for an ELISA format test, or for skin testing (skin prick tests are
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commonly used in allergy testing in pets and humans). Also, the auto-antibody test may be used in the detection of mite infections in a range of hosts.
DESCRIPTION OF THE INVENTION
As indicated above, the invention enables the detection of the scabies mite, Sarcoptes scabiei in the skin of various species of host mammals. The invention relies on the identification of certain mite molecules which are present in the skin during infection and which produce strong antibody responses in the host. One such molecule is the enzyme, alpha amylase, which is produced by mites. Another molecule is transferrin a normal blood constituent of the host that is apparently changed by the mite to stimulate the production of auto-antibodies which are characteristic of scabies infections. The detection assay is based on assays that use antibodies to detect the presence of the mite molecules or of host antibodies against these molecules. The immunoassay uses a reporter molecule bound to an antibody or another detection agent and this signals the presence of the mite by a colour change or other detectable event. The assay is more sensitive than current diagnostic techniques.
Thus, the invention uses one or more antigens or auto-antibodies, or a combination thereof, in various forms of detection assay including, but not limited to, an enzyme linked immunosorbent assay or other forms of detection assay. The assay will detect either, the presence of the antibody/antigen/auto-antibody in the skin or of antibody to the antigen(s) or auto-antibody in the serum of infected animals or man. The presence of the antibody/antigen/auto-antibody will be signalled by a colour change or other detectable event above a threshold level in an antigen or antibody detection assay. Animals giving a signal above this level will be deemed to be infected with Sarcoptes scabiei.
Methodology and Discussion of Results
Analysis of skin scrapings and ELISA
Our initial aim was to detect mite antigens in skin scrapings taken from pigs infected with Sarcoptes scabiei. A soluble protein extract of mange positive skin scrapings was made, and proteins separated using SDS-PAGE. The proteins were transferred to PVDF membrane, which was subsequently probed using sera from infected pigs. Detection of
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antigens was carried out using an anti Pig-HRP conjugate and chromogenic detection using DAB. We discovered through N-terminal amino acid sequencing that the majority of the soluble proteins in the extract were derived from pig serum, and not mites. The major proteins were serum albumin, immunoglobulin G (heavy and light chain), transferrin, haptoglobin and hemoglobin alpha-chain (see Figure 1). We also found that infected pigs produced auto-antibodies to these proteins, thus hampering our efforts readily to uncover a mite protein in pig skin scrapings. The identification of transferrin as one such immunoreactive antigen (80 kDa) prompted further investigation by ELISA.
We proceeded to test the reactivity of 50 pigs to transferrin in an ELISA format using serum albumin as a control, with the following protocol:
• Dilute antigen (transferrin; bovine source; SIGMA) in Carbonate coating buffer to a concentration of lOμg/ml and coat each well on a microlitre plate with 50μl, allow protein to bind overnight at 4°C.
• Flick plate and wash 3 times with water and block remaining sites with 250μl PBS (containing 0.05% Tween 20 and 0.25% BSA - used in all subsequent steps) for 1 hour at room temperature.
• Flick plate and wash 3 times with water. Add 50μl of sera from pigs diluted at various concentrations in PBS and incubate at 37°C for 1 hour.
• Wash plate (Flick plate and wash 3 times with water, add 200μl of PBS and let stand for 5 minutes, then wash 3 times with water).
• Add 50μl secondary antibody (Anti-Pig IgG Peroxidase Conjugate) diluted 1 :1000 in PBS, and incubate at 37°C for 1 hour. Wash plate as above.
• Add 50μl substrate (5mg in 10ml of Peroxide buffer) and read plate after 30 minutes at 405nm.
Analysis showed that antibody titer to the serum protein transferrin was higher in pigs infested with S. scabiei than uninfected pigs (Figure 2). The mean titre + S.D. of the negative pigs was determined to be 238 + 142. The mean titre + S.D. of the positive pigs was determined to be 1810 + 912. A positive cut off value of the mean negative pig value plus 2 S.D. was used. The percentage of positive pigs greater than this cut off value was 95%). The results revealed that the mange infected group of pigs had higher antibody titres to transferrin than the mange negative pigs. Bovine transferrin was shown to be 96.1%
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specific and 95% sensitive in differentiating the two sets of pigs in this preliminary study. This result is very interesting in view of the role of transferrin in the body and the underlying mechanism that results in the pig making auto-antibodies to such a ubiquitous molecule. Transferrin is the major serum glycoprotein that transports iron to most tissues in mammals (Steverding, et al. 1995). Thus, the host iron binding molecules are apparently important to mite metabolism.
Mite salivary protein α-amylase
In order to overcome the problem of the small amounts of recoverable mite antigen from
Sarcoptes scabiei and contamination with serum proteins, we decided to use alternate species of mite to produce a soluble protein extract. Dermatophagoides pteronyssinus and D. farinae (common house dust mite) free of any porcine serum proteins can be commercially sourced. We found that sera from pigs infected with S. scabiei reacted strongly to antigens from these dust mites in an ELISA test. The dust mite antigens also reacted in mange positive pigs in a skin based hypersensitivity assay. An extract was made, and the proteins were separated using SDS-PAGE and transferred onto PVDF membrane and probed.
We identified a protein with a molecular weight of 56 kDa that was only detected by serum of pigs after experimental infection with S. scabiei (Figure 3). The 56 kDa protein identified was excised from coomassie stained PNDF membrane and sequenced by Ν- terminal sequencing. The amino acid sequence of the 56 kDa protein was found to be DSK FSΝ PHF IGS RSN. Only one match (83%) was found in the SwissProt database (BLAST search). The identity of this protein was alpha-amylase (allergen Der P4) from D. pteronyssinus (Lake, et al., 1991; SwissProt accession number P49274). The published sequence for house dust mite alpha-amylase is K YXΝ PHF IGX RSN IT XLM E. Due to the low amount of alpha-amylase available from a Sarcoptes source, an alternate bacterial source of alpha-amylase was trialed. Purified alpha-amylase was obtained commercially (bacterial source, Bacillus species type II-A; SIGMA) and tested for reactivity to pigs by ELISA. In testing of a total of 44 pigs there was a difference in the reactivity of the two groups of pigs to alpha-amylase. The mean of the mange negative pigs + 2 S.D. gave an antibody cut off titre of 300. We found that the protein alpha-amylase was able to distinquish 100% of mange positive pigs from negative controls (Figure 4).
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In order to obtain larger quantities of an α-amylase related to the mite enzyme, an excretory/secretory preparation from the blowfly, Lucilia cuprina (Sandeman et al., 1990) was tested for activity. Enzyme activity was confirmed using a commercial substrate (Amylase 3, Sigma Diagnostics 577-3) and by reactivity with sera from mange infected pigs. The blowfly larval α-amylase was then isolated by extraction on Q Sepaharose (Pharmacia) and the amylase active fractions were pooled for ELISA testing. The ELISA was carried out as outlined above with the exception of two monoclonal antibodies substituting for the anti-pig IgG conjugate. These antibodies were mouse anti-porcine IgGl (Serotec, MCA635) and rat anti-mouse IgGl-HRP conjugate (MCA336P) and both were used at a 1 :2000 dilution. Pig serum samples used in the ELISA were obtained from abattoir slaughtered pigs diagnosed as having mange or other diseases (acute and chronic pneumonia, pleurisy, ileitis and pleuropneumonia) post mortem (Figure 5).
Method for preparation of dust mite extract
D. pteronyssinus mites were obtained commercially from CSL, Melbourne, Australia, while D. farinae mites were sourced from Allergon AB, Angelholm, Sweden. Extracts were prepared by placing 1 gram of mites in a 50ml plastic tube with 20ml of cold PBS. The tube was vortexed until a homogenous solution of mites and buffer was achieved, the tube was then placed on ice for 6 hours with periodic vortexing every 40 minutes. The homogenate was centrifuged for 10 minutes at 30,000g. The supernatant was aliquoted into 1ml volumes and stored at -70 °C.
Method of preparation of Lucilia cuprina larval excretion/secretion product (LESP) The larvae of L. cuprina were obtained from a laboratory colony maintained in the Parasite Control Laboratory, Department of Agriculture, La Trobe University. The flies were originally obtained from a natural infection of fine wool Merino sheep in southern Victoria, Australia. The colony was supplemented annually with wild-type flies from natural infections to prevent in-bred mutations. Crude LESP was prepared using a method described by Sandeman et al. (1990).
LESP was freeze dried, desalted to distilled water on a Sephadex G25 column (Pharmacia Biotech), freeze dried again and stored at -70°C. After reconstitution in DDW, two protease inhibitors (EDTA and PMSF at a final concentration of 1 mM) were added to prevent protein degradation.
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Method for partial purification of alpha-amylase from LESP using fast protein liquid chromatographv (FPLQ
Concentration and purification of the alpha-amylase activity of LESP was carried out on Fast flow Q Sepharose (Pharmacia Biotech). Q-Sepharose is strong anion exchanger. A pre-packed 5ml Q Sepharose fast flow column was equilibrated in buffer A (0.02M Tris- HCl buffer, pH 8.0). LESP was prepared by centrifugation (10 minutes at 30,000g) to remove any particulates, diluted in buffer A and filtered through a 0.22 m sterile filter (Millex GS; Millipore). The composition of the sample was adjusted to the pH and ionic strength of the starting buffer. The sample (10ml) was loaded onto the column in buffer A and protein elution was carried out using a linear gradient of buffer A to buffer B (0.02M Tris-HCl buffer containing 1M NaCl, pH 8.0). The elution gradient was 0-60% Buffer B over 40 minutes at a flow rate of 1ml per minute, then 60-100%) Buffer B over the next 20 minutes at the same flow rate. The elution profile was recorded by absorbance at 280nm. Alpha-amylase activity was determined using an amylase reagent (AMYLASE 3; Sigma Chemical Company).
Analysis of immunoreactive serine proteases in LESP
Further analysis was carried out on the semi-purified LESP fraction for other antigen/s of diagnostic use. SDS-PAGE and immunoblotting of the LESP fraction using two positive pigs (Figure 6, lanes B, C) and two negative pigs (Figure 6, lanes D, E) revealed several proteins which were bound by antibodies from positive pigs only. Two prominent proteins at 60 and 33 kDa were only identified by sera from the two positive individuals and not recognised by the two negative pigs in this preliminary study. N-terminal sequencing of the 60 and 33 kDa proteins in the benzamidine purified fraction gave the sequence shown in Table 1.
Table 1 Amino acid sequence of 60 and 33 kDa benzamidine sepharose purified proteins from LESP and homologous proteins identified from a BLAST search of the SwissProt. database.
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Comparing the 60 kDa sequence to the SwissProt. database (BLAST search) revealed the highest homology (71%) to two proteins. The first was Trypsin I from crayfish (Astacus astacus) (SwissProt accession number P00765, Titani, et al, 1983) and the second was Collagenolytic Protease 36 kDa precursor from crab (Paralithodes camtschatica) (SwissProt accession number P20734, Klimova, et al, 1990). Trypsin I is one of the five forms of the enzyme known to be present in crayfish. This protein is a serine protease that is more acidic than mammalian trypsin. Collagenolytic protease is a serine protease capable of degrading the native triple helix of collagen.
Analysis of the 33 kDa sequence to the SwissProt. database (BLAST search) revealed 65% homology to Collagenase Precursor (Hypodermin C) (Hypoderma lineatum) (SwissProt accession number P08897, Moire, et al, 1994). This enzyme is a serine protease (trypsin family) capable of degrading the native triple helix of collagen protein. It is isolated from the early cattle grub and is larval-specific.
Benzamidine sepharose chromatographv of LESP
Benzamidine Sepharose 6B (Pharmacia Biotech) which specifically binds trypsin and trypsin-like serine proteases, was prepared according to manufacturer's instructions. A
5ml column was prepared and equilibrated with binding buffer (50mM Tris-HCl pH 8.0).
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Freeze dried LESP was resuspended in 30ml distilled water and desalted on a G-25 column. The desalted LESP fraction was then run down the benzamidine column and the column was washed thoroughly in binding buffer. The bound proteins were eluted using 0.1M glycine-HCl pH 3.0. The bound fraction was immediately neutralised using 1M Tris pH 9.0. The eluted fraction was desalted and concentrated using a pressure-driven, disposable, ultrafiltration device for processing aqueous biological solutions (Ultrafree- PF; Millipore). After reconstitution in DDW, two protease inhibitors (EDTA and PMSF at a final concentration of 1 mM) were added to prevent protein degradation.
ELISA testing of the benzamidine fraction of LESP ELISA testing using the benzamidine purified fraction confirmed the apparent reactivity with mange positive pigs. It was evident that the benzamidine fraction of LESP was able to discriminate infected from non-infected individuals. The mean titre of the negative pigs was 533 + 206. The mean titre of positive pigs was 75 + 27. The cut off value (mean negative plus 2 S.D.) was 130. The percentage of positive pigs greater than this cut off value was 100%.
ELISA testing of trypsin
In light of the sequencing data and immunoreactivity of mange positive pigs to trypsin- like proteases in LESP, commercially available trypsin was trialed in ELISA. Testing of mange positive and negative pigs was carried out using a bovine source of the protein trypsin (Figure 7). It was shown that mange infected pigs had high immunoreactivity to trypsin. The mean titre of the negative pigs was 229 + 125. The mean titre of positive pigs was 1200 + 422. The cut off value (mean negative plus 2 S.D.) was 479. The percentage of positive pigs greater than this cut off value was 100%.
Validation of ELISA assay Trial design
Pig sera was examined from a drug efficacy trial consisting of pigs naturally infested with sarcoptic mites, divided into three groups. The first group (n=16) were left untreated. The second (n=12) and third (n=10) groups were treated with either Ivomectin® (IVOMEC, Merial) or Dectomax® (Pfizer) respectively. Pigs were weighed and examined and randomly allocated to one of the groups based on age, sex and mange lesions. Assessment
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of mange status for allocation into groups used ear lesion severity and calculation of a rubbing index.
A blood sample was collected prior to weaning (Day -2). On the day of weaning (Day 0) pigs in the treated groups were treated with either Ivermectin 1% (w/v) (IVOMEC S/C injection for swine, Merial) or Dectomax (Doramectin IM injection, Pfizer) at the rate of 1 ml/33 kg (300 mcg/kg). Pigs in the untreated group remained unmedicated. On Day 122, all pigs were bled again.
Calculating the numbers of mites on pigs
An area of approximately 2 cm2 on the left ear of all pigs was scraped and the material examined after KOH incubation (Dobson and Davies, 1992) for adult and immature mites. Scrapings were carried out on Day 0, 16 and 122. The total mite count for each pig, for each day of sampling, consisted of the sum of the adult and immature mites found.
Calculating ear lesions scores
The extent of ear lesions was determined using the following grading system. 0 = no lesion; 1 = lesion < 2 cm2; 2 = 2 cm2 but < 3 cm2; 3 = > 3 cm2 or multiple lesions.
Calculating the rubbing index of pigs
Between 25 and 50 pigs were selected for observation on the basis that they could be clearly seen from the observation position without having to move. The pigs were disturbed so that they all stood up. The number of rubbing episodes was counted over the next 15 minutes. The number of rubbing episodes was then divided by the number of pigs (Cargill and Dobson, 1979a). Pruritis is thought to be a better indicator of active S. scabiei infestation than the number of mites that can be found in skin scrapings, and has been used in monitoring the efficacy of acaricides in field studies (Courtney, et al, 1983).
Serological monitoring to confirm eradication of mange The antibody titres of pigs were monitored according to the ELISA method outlined previously using an alpha-amylase enriched fraction of LESP.
The mean total mite counts found in skin scrapings for the three groups was calculated from this raw data and is shown in Table 2.
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Table 2 Mean total mite counts from skin scrapings ± S.D. at Day 0, 16 and 122 for naturally infected pigs. Pigs were treated at Day 0 with either Ivermectin [T(I)] (n=12) or Dectomax [T(D)] (n=10), or left untreated (N) (n=16).
Serological monitoring of pigs using alpha-amylase from LESP
The antibody titres for the three groups of pigs is shown in Table 3 and Figure 8. All three groups showed similar low antibody titres at Day -2 with an average titre of 23. The antibody titres of all three groups at Day -2 were lower than that of the mange free herd comparison (C), which had a mean titre ± S.D. of 72 ± 26. At Day 122 the highest antibody titres were seen for the untreated group (N) with a mean titre ± S.D. of 600 ± 432. In comparison, the Ivermectin group [T(I)] was 225 ± 87 and the Dectomax group [T(D)] was 320 ± 103. It is important to note that the pigs tested at Day -2 were all weaners, and it is reported that pigs of this age do not produce high levels of antibodies to Sarcoptes.
Table 3 Mean antibody titre ± S.D. at Day 0 and 122 for naturally infected pigs. Pigs were either treated at Day 0 with Ivermectin (n=12) or Dectomax (n=10), or left untreated (n=16).
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Correlation of ELISA absorbance to mite numbers
The large standard deviation seen in the antibody titres of the untreated group (N) at Day 122 was investigated further. A correlation of the ELISA absorbance (1 in 800 dilution) for each pig was made to the total number of mites found in the individual's skin scrapings (Figure 9). A correlation value of R2 = 0.61 was obtained.
LEGENDS TO FIGURES
Figure 1. SDS-PAGE and staining of the porcine skin scraping protein extract. (A) 80 kDa= transferrin, (B) 69 kDa=serum albumin, (C) 55 kDa=Immunoglobin heavy chain, (D) 40kDa=haptoglobin, (E) 30 kDa=Immunoglobin light chain, (F) 14 kDa=hemoglobin. Molecular weight standards (mw) are also indicated in Kilodaltons (kDa).
Figure 2. Antibody levels to bovine transferrin in pigs infected with mange. ELISA testing was conducted using both mange free (n = 30) and mange positive sera (n = 20).
Figure 3. SDS-PAGE and immunoblotting of a crude Dermatophagoides farinae extract. The protein profile of the extract is shown in lanes A and B. Immunoblotting of the dust mite extract was carried out with positive pig sera for Sarcoptes infestation (D) and also with mange negative sera (E). A conjugate control where no primary antibody was tested is shown in lane F. Molecular weight markers (mw) are indicated in Kilodaltons (kDa).
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Figure 4. Reactivity of pig sera to alpha-amylase (bacterial source) as tested by ELISA. Two groups of pigs being either mange negative or positive were differentiated using a cut-off value of the mean titre of the mange negative pigs plus 2 standard deviations. All positive pigs tested had a value greater than this antibody cut-off titre value of 1/300.
Figure 5. Reactivity of abattoir pig sera to insect alpha-amylase as tested by ELISA. An alpha-amylase fraction was isolated from a Lucilia cuprina larval excretion/secretion product. Sera was obtained from pigs belonging to commercial piggeries that were diagnosed post mortem. Sera was classified into the following disease catagories; disease free, acute pneumonia, chronic pneumonia, pleurisy, and mange positive, The pigs in the pneumonia and pleurisy groups showed no clinical signs of mange post mortem.
Figure 6. Gel electrophoresis and immunoblotting of benzamidine purified Lucilia cuprina larval excretory/secretory product (LESP). The protein profile of the purified fraction is shown in lane A. Benzamidine purified LESP was transferred to PVDF membrane and immunoblotted with positive sera (B, C) and negative sera (D, E). Conjugate controls without primary antibody are shown in lane F. Molecular weight markers (mw) are indicated in Kilodaltons (kDa).
Figure 7. Reactivity of mange negative and positive pig sera to bovine trypsin. Antibody titres were monitored by ELISA.
Figure 8. ELISA analysis of serum antibodies to LESP alpha-amylase in pigs. Pigs from naturally infested sows were bled prior to weaning (Day -2) and at Day 122. Pigs in the treatment groups (T) were treated with acaricide at Day 0. N = untreated, T(I) = treatment group (Ivermectin), T(D) treatment group (Dectomax), C = mange free herd for comparison. Values shown are the mean antibody titres ± S. E.
Figure 9. Correlation of ELISA absorbance with total mite numbers in the untreated group (N) at Day 122 (cf Figure 8).
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Claims
1. A method for the detection of mite infection in an animal, including humans, in a sample taken from the animal, which method comprises contacting said sample with either: (a) an antibody/antibodies directed to, or that binds with: (i) one or more antigens in contact with the immune system of the animal early in said infection; or (ii) one or more antigens induced in the host animal by said infection (auto-antigen(s)), or (iii) a combination of said antigen(s) and auto-antigen(s), and detecting the binding to said sample of said antibody/antibodies to indicate the presence in said sample of said antigen(s) and/or auto-antigen(s); or (b) one or more antigens in contact with the immune system of the animal early in said infection, and detecting the binding to said sample of said antigen(s) to indicate the presence in said sample of an antibody/antibodies directed to, or that binds with said antigen(s), wherein the antigen(s) is selected from: a mite alpha-amylase, or a peptide with substantially the same amino acid sequence as a mite alpha-amylase, or an immunodominant region or epitope of a mite alpha-amylase or a peptide with substantially the same amino acid sequence thereof; a mite trypsin, or a peptide with substantially the same amino acid sequence as a mite trypsin, or an immunodominant region or epitope of a mite trypsin or a peptide with substantially the same amino acid sequence thereof; a mite trypsin-like protease, or a peptide with substantially the same amino acid sequence as a mite trypsin-like protease, or an immunodominant region or epitope of a mite trypsin- like protease or a peptide with substantially the same amino acid sequence thereof; a mite serine protease, or a peptide with substantially the same amino acid sequence as a mite serine protease, or an immunodominant region or epitope of a mite serine protease or a peptide with substantially the same amino acid sequence thereof; and the excretory/secretory product (ESP) of an arthropod, or a combination thereof; and wherein the auto-antigen(s) is selected from transferrin, haptoglobin or other serine protease homolog or serine-like protease, or a combination thereof.
Substitute sheet
2. The method of claim 1 wherein said sample is contacted with an antibody/ antibodies directed to, or that binds with: (i) one or more antigens in contact with the immune system of the animal early in said infection; or (ii) one or more antigens induced in the host animal by said infection (auto-antigen(s)), or (iii) a combination of said antigen(s) and auto-antigen(s), and detecting the binding to said sample of said antibody/antibodies to indicate the presence in said sample of said antigen(s) and/or auto- antigen(s).
3. The method of claim 1 or 2 wherein said sample is contacted with an antibody/ antibodies directed to, or that binds with one or more antigens in contact with the immune system of the animal early in said infection, and detecting the binding to said sample of said antibody/antibodies to indicate the presence in said sample of said antigen(s).
4. The method of claim 1 or 2 wherein said sample is contacted with an antibody/ antibodies directed to, or that binds with one or more antigens induced in the host animal by said infection (auto-antigen(s)), and detecting the binding to said sample of said antibody/antibodies to indicate the presence in said sample of said auto-antigen(s).
5. The method of any one of claims 1 to 4 wherein said antibody/antibodies is a monoclonal antibody.
6. The method of any one of claims 1 to 5 wherein said sample is a skin scraping or skin washing or ear wax of the animal.
7. The method of claim 1 wherein said sample is contacted with one or more antigens in contact with the immune system of the animal early in said infection, and detecting the binding to said sample of said antigen(s) to indicate the presence in said sample of an antibody/antibodies directed to, or that binds with said antigen(s).
8. The method of claim 1 or 7 wherein said sample is serum or a skin scraping or skin washing of the animal.
Substitute sheet
9. The method of any one of claims 1 to 8 wherein the antigen is an alpha-amylase or an immunodominant region or epitope of an alpha-amylase.
10. The method of claim 9 wherein the alpha-amylase is alpha-amylase of Sarcoptes scabiei.
5 11. The method of claim 9 wherein the alpha-amylase is alpha-amylase of Dermatophagoides pteronyssinus or D. farinae.
12. The method of claim 9 wherein the alpha-amylase is alpha-amylase of Lucilia cuprina.
13. The method of any one of claims 1 to 8 wherein the antigen is a trypsin or an 0 immunodominant region or epitope of a trypsin.
14. The method of claim 13 wherein the trypsin is trypsin of Sarcoptes scabiei.
15. The method of claim 13 wherein the trypsin is trypsin of Dermatophagoides pteronyssinus or D. farinae.
16. The method of claim 13 wherein the trypsin is trypsin of Lucilia cuprina.
5 17. The method of any one of claims 1 to 8 wherein the antigen is a trypsin-like protease or an immunodominant region or epitope of a trypsin-like protease.
18 The method of claim 17 wherein the trypsin-like protease is trypsin-like protease of Sarcoptes scabiei.
19. The method of claim 17 wherein the trypsin-like protease is trypsin-like protease of Q Dermatophagoides pteronyssinus or D. farinae.
Substitute sheet
20. The method of claim 17 wherein the trypsin-like protease is trypsin-like protease of Lucilia cuprina.
21. The method of any one of claims 1 to 8 wherein the antigen is a serine protease or an immunodominant region or epitope of a serine protease.
5 22. The method of claim 21 wherein the serine protease is serine protease oϊ Sarcoptes scabiei.
23. The method of claim 21 wherein the serine protease is serine protease of Dermatophagoides pteronyssinus or D. farinae.
24. The method of claim 21 wherein the serine protease is serine protease oϊ Lucilia Q cuprina.
25. The method of claim 1 wherein the antigen is excretory/secretory product (ESP) of Lucilia cuprina or other such arthropod.
26. The method of claim 1 or 25 wherein the antigen is larval excretory/secretory product (LESP) oϊ Lucilia cuprina.
5 27. The method of any one of claims 1 , 2, 4 to 6 or 9 to 26 wherein the auto-antigen is transferrin.
28. The method of any one of claims 1, 2, 4 to 6 or 9 to 26 wherein the auto-antigen is haptoglobin.
29. The method of any one of claims 1, 2, 4 to 6 or 9 to 26 wherein the auto-antigen is Q a serine protease homolog or serine-like protease.
Substitute sheet
30. The method of any one of claims 1 to 29 wherein the binding to said sample is detected by an ELISA testing or fluorescent/luminol binding assay or radioimmunoassay, or precipitation or agglutination.
31. The method of claim 30, wherein the binding to said sample is detected by an 5 ELISA test.
32. The method of claim 31, wherein the binding to said sample is detected by an ELISA test specific to Sarcoptes scabiei.
33. The method of any one of claims 1 to 29 wherein the binding to said sample is detected by a skin based hypersensitivity test.
10 34. The method of claim 33 wherein the binding to said sample is detected by a skin test specific to Sarcoptes scabiei.
35. The method of any one of claims 1 to 34 wherein the animal is selected from pigs, horses, cows, sheep, dogs, cats, wombats, buffalo, deer, rabbits, donkeys, goats, foxes, camels and humans.
15 36. A diagnostic test kit for detection of mite infection in an animal, including humans, comprising a device for contacting and binding a sample taken from the animal, and a detection means for communicating with the sample, wherein the detection means comprises either:
(a) an antibody/antibodies directed to, or that binds with: (i) one or more antigens in 20 contact with the immune system of the animal early in said infection; or (ii) one or more antigens induced in the host animal by said infection (auto-antigen(s)), or (iii) a combination of said antigen(s) and auto-antigen(s), wherein said antigen(s) and/or auto-antigen(s) is defined in any one of claims 1 or 9 to 29, together with a reagent(s) that is responsive to a reaction between said antibody/antibodies with 25 said antigen(s) and/or auto-antigen(s); or
Substitute sheet
(b) one or more antigens in contact with the immune system of the animal early in said infection as defined in any one of claims 1 or 9-29, together with a reagent(s) that is responsive to a reaction between said antigen(s) and an antibody/antibodies directed to, or that binds with said antigen(s).
5 37. The diagnostic test kit of claim 36, wherein the detection means comprises an antibody/antibodies directed to, or that binds with: (i) one or more antigens in contact with the immune system of the animal early in said infection; or (ii) one or more antigens induced in the host animal by said infection (auto-antigen(s)), or (iii) a combination of said antigen(s) and auto-antigen(s), wherein said antigen(s) and/or auto-antigen(s) is defined in 10 any one of claims 1 or 9 to 29, together with a reagent(s) that is responsive to a reaction between said antibody/antibodies with said antigen(s) and/or auto-antigen(s).
38. The diagnostic test kit of claim 36 or 37, wherein the antibody/antibodies is directed to, or binds with one or more antigens in contact with the immune system of the animal early in said infection.
15 39. The diagnostic test kit of claim 36 or 37, wherein the antibody/antibodies is directed to, or binds with one or more antigens induced in the host animal by said infection (auto-antigen(s)).
40. The diagnostic test kit of any one of claims 36 to 39 wherein said antibody/ antibodies is a monoclonal antibody.
20 41. The diagnostic test kit of any one of claims 36 to 40 wherein said sample is a skin scraping or skin washing or ear wax of the animal.
42. The diagnostic test kit of claim 36 wherein the detection means comprises one or more antigens in contact with the immune system of the animal early in said infection, together with a reagent(s) that is responsive to a reaction between said antigen(s) and an 25 antibody/antibodies directed to, or that binds with said antigen(s).
Substitute sheet
43. The diagnostic test kit of claim 36 or 42 wherein said sample is serum or a skin scraping or skin washing of the animal.
44. The diagnostic test kit of any one of claims 36 to 43, wherein the antigen is as defined in any one of claims 9 to 12.
45. The diagnostic test kit of any one of claims 36 to 43 wherein the antigen is as defined in claim 10.
46. The diagnostic test kit of any one of claims 36 to 43, wherein the antigen is as defined in any one of claims 13 to 16.
47. The diagnostic test kit of any one of claims 36 to 43, wherein the antigen is as defined in claim 14.
48. The diagnostic test kit of any one of claims 36 to 43, wherein the antigen is as defined in any one of claims 17 to 20.
49. The diagnostic test kit of any one of claims 36 to 43, wherein the antigen is as defined in claim 18.
50. The diagnostic test kit of any one of claims 36 to 43, wherein the antigen is as defined in any one of claims 21 to 24.
51. The diagnostic test kit of any one of claims 36 to 43, wherein the antigen is as defined in claim 22.
52. The diagnostic test kit of any one of claims 36 to 43, wherein the antigen is as defined in claim 25 or 26.
53. The diagnostic test kit of any one of claims 36 to 43, wherein the antigen is as defined in claim 26.
Substitute sheet
54. The diagnostic test kit of any one of claims 36, 37 or 39 to 41, wherein the auto- antigen is as defined in any one of claims 27 to 29.
55. The diagnostic test kit of claim 54, wherein the auto-antigen is as defined in claim 27.
56. The diagnostic test kit of any one of claims 36 to 55 wherein the reaction is detected by an ELISA testing or fluorescent/luminol binding assay or radioimmunoassay, or precipitation or agglutination.
57. The diagnostic test kit of claim 56, wherein the reaction is detected by an ELISA test.
58. The diagnostic test kit of claim 57, wherein the reaction is detected by an ELISA test specific to Sarcoptes scabiei.
59. The diagnostic test kit of any one of claims 36 to 55 wherein the reaction is detected by a skin based hypersensitivity test.
60. The diagnostic test kit of claim 59 wherein the reaction is detected by a skin test specific to Sarcoptes scabiei.
61. The diagnostic test kit of any one claims 36 to 60 wherein the animal is selected from pigs, horses, cows, sheep, dogs, cats, wombats buffalo, deer, rabbits, donkeys, goats, foxes, camels and humans.
62. A diagnostic reagent for detection of mite infection in an animal, including humans, comprising an antibody/antibodies directed to, or that binds with: (i) one or more antigens in contact with the immune system of the animal early in said infection; or (ii) one or more antigens induced in the host animal by said infection (auto-antigen(s)), or (iii) a combination of said antigen(s) and auto-antigen(s), wherein said antigen(s) and/or auto-
Substitute sheet
antigen(s) is as defined in any one of claims 1 or 9 to 29, and said antibody/antibodies is/are labelled so as to permit detection of the reaction between said antibody/antibodies with said antigen(s) and/or auto-antigen(s).
63. The diagnostic reagent of claim 62 wherein the antibody/antibodies is directed to, or binds with one or more antigens in contact with the immune system of the animal early in said infection.
64. The diagnostic reagent of claim 62 wherein the antibody/antibodies is directed to, or binds with one or more antigens induced in the host animal by said infection (auto- antigen(s)).
65. The diagnostic reagent of any one of claims 62 to 64 wherein said antibody/ antibodies is a monoclonal antibody.
66. The diagnostic reagent of any one of claims 62 to 65, wherein the antigen is as defined in any one of claims 9 to 12.
67. The diagnostic reagent of any one of claims 62 to 65 wherein the antigen is as defined in claim 10.
68. The diagnostic reagent of any one of claims 62 to 65, wherein the antigen is as defined in any one of claims 13 to 16.
69. The diagnostic reagent of any one of claims 62 to 65, wherein the antigen is as defined in claim 14.
70. The diagnostic reagent of any one of claims 62 to 65, wherein the antigen is as defined in any one of claims 17 to 20.
71. The diagnostic reagent of any one of claims 62 to 65, wherein the antigen is as defined in claim 18.
Substitute sheet
72. The diagnostic reagent of any one of claims 62 to 65, wherein the antigen is as defined in any one of claims 21 to 24.
73. The diagnostic reagent of any one of claims 62 to 65, wherein the antigen is as defined in claim 22.
74. The diagnostic reagent of any one of claims 62 to 65, wherein the antigen is as defined in claim 25 or 26.
75. The diagnostic reagent of any one of claims 62 to 65, wherein the antigen is as defined in claim 26.
76. The diagnostic reagent of any one of claims 62 or 64 to 75, wherein the auto- antigen is as defined in any one of claims 27 to 29.
77. The diagnostic reagent of claim 76, wherein the auto-antigen is as defined in claim
27.
78. The diagnostic reagent of any one of claims 62 to 75, wherein the detection is by an ELISA testing or fluorescent/luminol binding assay or radioimmunoassay, or precipitation or agglutination.
79. The diagnostic reagent of claim 78, wherein the detection is by an ELISA test.
80. The diagnostic reagent of claim 79, wherein the detection is by an ELISA test specific to Sarcoptes scabiei.
81. The diagnostic reagent of any one of claims 62 to 75 wherein the detection is by a skin based hypersensitivity test.
Substitute sheet
82. The diagnostic reagent of claim 81 wherein the detection is by a skin test specific to Sarcoptes scabiei.
83. An antibody/ antibodies directed to, or that binds with: (i) one or more antigens; or (ii) one or more auto-antigen(s), or (iii) a combination of said antigen(s) and auto- antigen(s), as defined in any one of claims 1 or 9 to 29.
84. The antibody of claim 83, which is a monoclonal antibody that binds to said antigen or auto-antigen.
Substitute sheet
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU62539/00A AU6253900A (en) | 1999-08-09 | 2000-08-04 | Method for detection of mite infection |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AUPQ2076 | 1999-08-09 | ||
| AUPQ2076A AUPQ207699A0 (en) | 1999-08-09 | 1999-08-09 | Method for detection of mite infection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2001011365A1 true WO2001011365A1 (en) | 2001-02-15 |
Family
ID=3816256
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/AU2000/000930 WO2001011365A1 (en) | 1999-08-09 | 2000-08-04 | Method for detection of mite infection |
Country Status (2)
| Country | Link |
|---|---|
| AU (1) | AUPQ207699A0 (en) |
| WO (1) | WO2001011365A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP1920239A2 (en) * | 2005-07-27 | 2008-05-14 | Cytosignet, Inc. | Detection and measurement of blood-feeding activity |
| CN107290535A (en) * | 2017-08-09 | 2017-10-24 | 四川农业大学 | The application of itch mite inorganic pyrophosphatase and the kit for diagnosing psoroptic mange |
| CN107478835A (en) * | 2017-08-09 | 2017-12-15 | 四川农业大学 | The application of itch mite protein tyrosine kinase and the kit for diagnosing psoroptic mange |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997042318A1 (en) * | 1996-05-02 | 1997-11-13 | The Minister Of Agriculture Fisheries And Food | Vaccines |
| GB2327346A (en) * | 1997-07-04 | 1999-01-27 | Univ Melbourne | Parasite vaccine |
-
1999
- 1999-08-09 AU AUPQ2076A patent/AUPQ207699A0/en not_active Abandoned
-
2000
- 2000-08-04 WO PCT/AU2000/000930 patent/WO2001011365A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997042318A1 (en) * | 1996-05-02 | 1997-11-13 | The Minister Of Agriculture Fisheries And Food | Vaccines |
| GB2327346A (en) * | 1997-07-04 | 1999-01-27 | Univ Melbourne | Parasite vaccine |
Non-Patent Citations (4)
| Title |
|---|
| E.L. WOOTEN AND S.M. GAAFAR: "Detection of serum antibodies to sarcoptic mange mite antigens by the passive hemagglutination assay in pigs infected with sarcoptes cabiei var. suis", VETERINARY PARASITOLOGY (ELSEVIER SCIENCE, AMSTERDAM), vol. 15, September 1984 (1984-09-01), pages 309 - 316 * |
| P. WALLGREN AND S. BORNSTEIN: "Serodiagnosis of sarcoptic mange in pigs", VETERINARY RECORD, vol. 141, 5 July 1997 (1997-07-05), pages 8 - 12 * |
| P. WALLGREN AND S. BORNSTEIN: "The spread of porcine sarcoptic mange during the fattening period revealed by development of antibodies to sarcoptes scabiei", VETERINARY PARASITOLOGY (ELSEVIER SCIENCE, AMSTERDAM), vol. 73, December 1997 (1997-12-01), pages 315 - 324 * |
| W. HOLLANDERS ET AL.: "Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of sarcoptic mange in swine", VETERINARY PARASITOLOGY (ELSEVIER SCIENCE, AMSTERDAM), vol. 69, April 1997 (1997-04-01), pages 117 - 123 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1920239A2 (en) * | 2005-07-27 | 2008-05-14 | Cytosignet, Inc. | Detection and measurement of blood-feeding activity |
| EP1920239A4 (en) * | 2005-07-27 | 2009-03-25 | Cytosignet Inc | Detection and measurement of blood-feeding activity |
| US7727734B1 (en) | 2005-07-27 | 2010-06-01 | Cytosignet, Inc. | Detection and measurement of blood-feeding activity |
| EP2357476A3 (en) * | 2005-07-27 | 2012-03-21 | Cytosignet, Inc. | Detection and measurement of blood-feeding activity |
| US8460890B2 (en) | 2005-07-27 | 2013-06-11 | Cytosignet, Inc. | Detection and measurement of blood-feeding activity |
| US8877451B2 (en) | 2005-07-27 | 2014-11-04 | Cytosignet, Inc. | Detection and measurement of blood-feeding activity |
| CN107290535A (en) * | 2017-08-09 | 2017-10-24 | 四川农业大学 | The application of itch mite inorganic pyrophosphatase and the kit for diagnosing psoroptic mange |
| CN107478835A (en) * | 2017-08-09 | 2017-12-15 | 四川农业大学 | The application of itch mite protein tyrosine kinase and the kit for diagnosing psoroptic mange |
| CN107290535B (en) * | 2017-08-09 | 2019-03-08 | 四川农业大学 | The application of itch mite inorganic pyrophosphatase and the kit of diagnosis psoroptic mange |
| CN107478835B (en) * | 2017-08-09 | 2019-03-08 | 四川农业大学 | The application of itch mite protein tyrosine kinase and the kit of diagnosis psoroptic mange |
Also Published As
| Publication number | Publication date |
|---|---|
| AUPQ207699A0 (en) | 1999-08-26 |
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