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WO2015035474A1 - Test sérologique - Google Patents

Test sérologique Download PDF

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Publication number
WO2015035474A1
WO2015035474A1 PCT/AU2014/050231 AU2014050231W WO2015035474A1 WO 2015035474 A1 WO2015035474 A1 WO 2015035474A1 AU 2014050231 W AU2014050231 W AU 2014050231W WO 2015035474 A1 WO2015035474 A1 WO 2015035474A1
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Prior art keywords
seq
amino acid
acid sequence
protein
antibody
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English (en)
Inventor
Glenn Francis Browning
Philip Francis MARKHAM
Wasala Mudiyanselage Nadeeka Kumari WAWEGAMA
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University of Melbourne
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University of Melbourne
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Priority claimed from AU2013903549A external-priority patent/AU2013903549A0/en
Application filed by University of Melbourne filed Critical University of Melbourne
Publication of WO2015035474A1 publication Critical patent/WO2015035474A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0241—Mollicutes, e.g. Mycoplasma, Erysipelothrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/30—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56933—Mycoplasma

Definitions

  • the present specification teaches a serological test for Mycoplasma and the development of prophylactic and therapeutic compositions to treat ruminant subjects infected with or potentially exposed to Mycoplasma species.
  • Mycoplasma hovis i the most frequently isolated aetiological agent in naturally occuning outbreaks of bovine respiratory disease (BRD) in veal ealves (Gagea .ei al. (2006) J. Vet, Diagii. Invest 18:29-40; Arcangioli el at. (2008) Vet. J. 177:89-93), a chronic disease characterized by pneumonia and arthritis.
  • Mycoplasma bovis is also responsible for outbreaks of keratoconjunctivitis (A!berti et ai. (2006) J, Vet. Diagn,. Invest. /i?:41-51), mastitis (Hale et al.
  • Vsps variable surface proteins
  • Hsp60 and P48 (Scherm et al (2002) Vet. Microbiol #0: 1.41-150; Robino et ai (2005) Vet. Microbiol 109:201-209).
  • these are not ideal antigens for immunodiagnosis, because of the phase and antigenic variatio seen in Vsps, and the similarities between Hsp60 and P48 and their orthologs in the closely related species such as M. ag k tiae. Therefore, there remains a need to identify a better Mycoplasma antigen for serological diagnosis.
  • An immunogenic protein is identified useful in the identification of Mycoplasm spp and in the development of prophylactic and therapeutic compositions to manage Mycopimma infection and exposure.
  • the immunogenic protein is referred to herein as Mycopimma immunogenic lipase A (MilA).
  • MoilA Mycopimma immunogenic lipase A
  • Reference to. "MilA” includes the MilA protein from Mycopimma bovis and homologs of MilA from othe Mycopimma spp as well as antibody-binding fragments of MilA.
  • Region 1 of MilA is defined by the consensus sequence set forth i SEQ ID NOT, Examples of region 1 of MilA include MilA from M, bovis FG45 strain (type strain) [SEQ ID NG:2], and MilA homologs from M. bovis strain Hubei-1 (SEQ ID NQ:3), Mycoplasma agalacfia strain PG2 (SEQ ID NO: 4), Mycoplasma colimbin m strain SF7 (SEQ ID NO: 5). Mycoplasma fermantan strai JER (SEQ ID NO:6), Mycoplasma pulmonis (SEQ ID NO: 7) and Mycoplasma hyopneumomae (SEQ ID NO:S).
  • Full length MilA is defined in SEQ ID ' NO: 5 (M hovis PG45), SEQ ID NO: 16 ( btnis Hubei-1 ), SEQ ID NO: 1.7 (M agalacPae PG2), SEQ ID NO: 18 (M columbima SF7), SEQ ED NO: 1.9 (M, fermantans 1E ), SEQ ID G:20 ( pulmonis) and SEQ ID ⁇ O.2 i (M. hyopn umoniae).
  • the nucleotide sequence encoding MilA optimized for expression in E. coll is set forth in SEQ ID NO; 13.
  • the genomic nucleotide sequence from M. bovis PG45 is set forth in SEQ ID NO: 14.
  • a method for detecting current or prior exposur of a ruminant subject to a species of Mycoplasma comprising contacting an antibody containing sample from the subject with an immobilized protein or derivative thereof comprising the amino acid sequence set forth in SEQ ID NO: l or an amino acid sequence having at least 40% similarity to SEQ ID NOT after optimal alignment or an antibody-binding fragment of the protein, for a time and under conditions sufficient for an antibody specific for the protein, if present in the sample, to bind to the protein and then detecting tor the presence of bound antibody wherein the presence of bound antibody is indicative of current or prior exposure to Mycoplasma spp.
  • (OOlOj Further enabled is a method for detecting current or prior exposure of a raminant subject to a species of Mycoplasma, the method comprising contacting an antibody containing sample from the subject with an immobilized protein or derivative thereof comprising the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence having at least 40% similarity to SEQ ID NO: 2 after optimal alignment or an antibody-binding fragment of the protein, for a time and under condi tions sufficient for an antibody specific for the protein, if present in the sample, to bind to the protein and then detecting for the presence of bound antibod wherein the presence of bound antibody is indicative of current or prior exposure to Mycoplasma spp.
  • [0011 J Taught herein is a method for detecting current or prior exposure of a ruminant subject to a species of Mycoplasma, the method comprising contacting an antibody containing sample from the subject with an immobilized protein or derivative thereof comprising the amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence having at least 40% similarity to SEQ ID NO: 15 after optimal alignment or an antibody- binding fragment of the protein, for a time and under conditions sufficient for an antibody specific for the protein, if present in the sample, to bind to the protein and then detecting for the presence of bound antibody wherein the presence of bound antibody is indicative of current or prior exposure to Mycoplasma spp,
  • the present specification teaches a device for screening for current or prior exposure of a. subject to a species of Mycoplasma, the device comprising a immobilized protein or derivative thereof comprising the amino acid sequence set forth in SEQ ID NO: I or an amino acid sequence having at least 40% similarity to SEQ ID NO: I after optimal alignment or an antibody-binding fragment of the protein, the device further comprising means to contact a sample f om the subject with the immobilized protein.
  • Enabled herei is a device for screening for current or prior exposure of a subject to a species of Mycoplasma, the device comprising an immobilized protein or derivative thereof comprising the amino acid sequence set forth in SEQ ID NO:2 or an amino acid .sequence having at least 40% similarity to SEQ ID O:2 after optimal alignment or an antibody-binding fragment of the protein, the device further comprising means to contact a sample from the subject with the immobilized protei .
  • a device for screening for current or prior exposure of a subject to a species of Mycoplasma comprising an immobilized protein or derivative thereof comprising the amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence having at least 40% similarity to SEQ ID NO: 15 after optimal alignment or an antibody-binding fragment of the protein, the device further comprising means to contact a sample from the subject with the immobilized protein.
  • Taught herein is a method for controlling infection by a species of Mycoplasma in a ruminant subject, the method comprising administering to the subject an antibody- inducing effective amount of a protein or derivative thereof comprising the amino acid sequence set forth in SEQ ID NO: l or an amino acid sequence having at least 40% similarity to SEQ ID NO: l after optimal alignment for a time and under conditions sufficient to generate antibodies to said protein.
  • Enabled herein is a method for controlling infection by a species of Mycoplasm or a condition caused or exacerbated by infection with Mycoplasma in a ruminant subject, the method comprising administering to the subject an antibody-inducing effective amount of a protein or derivative thereof comprising the amino acid sequence set forth in SEQ ID NO:2 or an amino aci d sequence having at least 40% similarity to SEQ ID NO:2 after optimal alignment for a time and under conditions sufficient to generate antibodies to the protein.
  • Infection by Mycoplasma spp ca lead to bovine respiratory disease, keratoconjunctivits, mastitis, meningitis, otitis media, and genital tract disease.
  • Taught herein is a method for controlling infection by a species of Mycoplasma or a condition caused or exacerbated by infection with Mycoplasma in a ruminant subject, the method comprising administering to the subject a antibody-inducing effective amount of a protein or derivative thereof comprising the amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence having at least 40% similarity to SEQ ID NO.1 5 after optimal alignment for a time and under conditions sufficient to generate antibodies to the protein.
  • Infectio by Mycoplasma spp can lead to bovine respiratory disease, keratoconjunctivits, mastitis, meningitis, otitis media, and genital tract disease.
  • composition comprising a protein or derivative thereof campnsin the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 40% similarity to SEQ ID NO: l after optimal alignment and one or more pharmaceutically acceptable carriers, excipients and/or diluents.
  • composition comprising a protein or derivative thereof comprising the amino acid sequence set forth in SEQ ID NG:2 or an amino acid sequence having at least 40% similarity to SEQ ID NQ:2 after optimal alignment and one or more pharmaceutically acceptable carriers, excipients and/or diluents,
  • Taught herein is a composition comprising a protein or derivative thereof comprising the amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence having at least 40% similarity to SEQ ID NO: 15 after optimal alignment and one or more pharmaceutically acceptable carriers, excipients and/or diluents,
  • the protein comprises an amino acid sequence selected from the listing consisting of SEQ ID NOs:2 through 8 or is an antibody-binding fragment thereof or comprises an amino acid sequence having at least 40% similarit to any one of SEQ ID N s.2 through 8 after optimal alignment.
  • the protein comprises an amino acid sequence selected from the listing consisting of SEQ ID NOs: 15 through 21 or is an antibody-binding fragment thereof or comprises an amino acid sequence having at least 40% similarity to an one of SEQ ID NOs: 15 through 21 after optimal alignment,
  • the protein comprises the amino acid sequence set forth in SEQ ID NO:2 or is an antibody-binding fragment thereof.
  • the protein comprises the amino acid sequence set forth in SEQ ID NO: 15 through 21 or is an antibody-binding fragment thereof.
  • the ruminant animal is selected from the list consisting of cow, sheep, goat, giraffe, yak, camel, llama, antelope and macropod.
  • the Mycoplasm is Mycoplasma bmis and the ruminant animal is a cow.
  • SEQ ID NO Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO).
  • the SEQ ID NOs correspond numerically to the sequence identifiers ⁇ 400> 1 (SEQ ID NO:l), ⁇ 400>2 (SEQ ID NO: 2), etc.
  • a summary of the sequence identifiers is provided in Table 1.
  • a sequence listing is provided after the claims.
  • Figure 1 is a diagrammatical representation of a 6 kbp region of MBQVPG45J 10 (Mil A) gene cloned as two 3 kbp fragments, AB and CD, and subcloned as four 1 ,5 kbp DNA fragments, A, B, C and D, with the expressed gene sequences bounded by restriction endonuclease cleavage sites.
  • Figure 2 is a ph.otographi.cal representation of Western blots of whole cell proteins of bovis strain 3683 probed with sera from 6 calve (A-F) collected on: 1, day 0; 2, day 24. Uninfected, calves inoculated with Mycoplasma culture medium; Infected, calves inoculated with M hovis strain 3683; Arrow, protein of interest seen in strip 2 in infected calves,
  • Figure 3 is a photographical representation of SDS-P GE and Western blot of Mil A (226 kDa). Lanes: 1, Spectra (Trade Mark) Multicolor high range protein ladder (Fermentas); 2, whole cell proteins of M. hovis strain 3683 stained with Coomassie blue; 3, whole cell proteins of M agaiactkie strain PG2 stained with Coomassie blue; 4, Western blot of whole cell proteins of M bovis strain 3683 probed with M fows-specific calf sera; 5, Western blot of whole cell proteins of , agaiactkie strain PG2 probed with . bovis- specific calf sera,
  • Figures 4(i) and (ii) are photographical representations showing expression of recombinant proteins in E. coli JM 109 cells, (i) Whole cell proteins of induced clones separated in a 12% w/v polyacryl mide gel stained with Coomassie blue, (ii ) transferred to a PVDF membrane and probed with pooled M, ⁇ , ⁇ -specific bovine sera. Lanes: 1 , PageRuler prestained protein ladder (Thermo Scientific); 2, pGEX-4T-l-w?
  • Figure 5 is a diagrammatical representation showing physical map of genes in the region surrounding MBOVPG45_07I0 (mi LA). The predicted size of the gene product is shown as the number of amino acids (aa). LP, lipoprotein; rpoB, DNA-directed RNA polymerase subrjnit beta; rpoC, DNA-directed RNA polymerase subunit beta; BQVPG45_0710 (mi/A), membrane protein; HP, hypothetical protein; ISMbov 7, transposase truncated protein; ISMbov 6, M42 family glutamyl aminopeptidase.
  • FIG. 6 is a diagrammatical representation showing multiple sequence alignment of region 1 of mycoplasma immunogenic lipase (MtlA) with bomo!ogs in other mycoplasmas and the mycoplasma GDSL carboxyesterases,
  • Figure 7 is a graphical representation showing release of resorufm by the lipase activity of GST-MilA-ab and GST-p65 over 30 min, with GST as the negative control.
  • Figures 8(i) and (ii) are graphical representations showing Logio IgG antibody titers of individual calves on day (i) 1.7 and (ii) day 24 after infection in. Experiment 1. The bar indicates the mea of each group.
  • Figures 9(i) and (it) are graphical representations showing Logio IgG antibody titers of individual calves on (i) day 17 and (ii) day 24 after infection in Experiment 2, The bar indi cates the mean of each group.
  • Figure 10 is a graphical representation of mea ⁇ /. bovis- specific IgM antibody titers of each group in Experiment 3. Bars indicate standard deviation,
  • Figure 11 is a graphical representation of mean M. ovis- specific IgM antibody titers of each group in Experiment 3. Bars indicate standard deviation,
  • Figure 12 is a schematic diagram of the piasmid vector pGEX-4T-l (reproduced from the GE Healthcare pGEX vector map).
  • Figure 13 is a representation of physical maps of mycoplasma hpmologues of MBOVPG45_710 (milA) and adjacent genes. Homologous proteins are shaded the same colour. Numbers above the genes indicate the length of the predicted product in amin acids.
  • MBOVPG45 710 membrane protein
  • MMB_0654 conserved hypothetical transmembrane protein * MAGa6830, conserved hypothetical protein
  • MAG_6100 conserved hypothetical protein
  • MMB__03 I 8 conserved hypothetical protein
  • MFE_02570 MfeM64YMschreib 0307 lipase/hypothetical protein
  • CiMl endo-l,4-beta-glucanase; ⁇ ⁇ , DMA topoisomerase; vvrA t uvrABC system protei A; vpmaYl, variable surface lipoprotein Y; LicA, lichenan-specifie ⁇ component.
  • Figure 14 is a graphical representation of M. fev/ -specific IgG ELISA results for sera from feedlot cattle. DETAILED DESCRIPTION
  • the instant specification enables an isolated protein or an antibody-binding fragment thereof comprising an amino acid sequence set forth i n SEQ ID NO: l or an amino acid sequence having at least 40% similarity to SEQ ID NO: ! after optimal alignment.
  • Reference to "at least 40%" means 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 ? 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, .89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100% similarity to a recited sequence identifier.
  • the protein or a derivative thereof is from Mycoplasma hovis type strain PG45 and comprises the amino acid sequence as set forth in SEQ ID NO;2 or an. amino acid sequence having at least 40% similarity to SEQ ID NO:2 after optimal alignment.
  • the protein or a derivative thereof is from Mycoplasma /> i v.v type strain PG45 and comprises the amino acid sequence as set forth in SEQ ID NO; 15 or an amino acid sequence having at least 40% similarity to SEQ ID NO: 15 after optimal alignment.
  • the protei or a derivative thereof is from Mycoplasma bovis strain Hubei-1 and comprises amino acid sequence as set forth in SEQ ID NO: or an amino acid sequence having at least 40% similarity to SEQ ID NO; 3 after optimal alignment,
  • the protein or a derivative thereof is from Mycoplasma agalactia strain PG2 and comprises amino acid sequence as set forth in SEQ ID NQ;4 or an amino acid sequence having at least 40% similarity to SEQ ID NO:4 after optimal alignment,
  • the protein or a derivative thereof is from Mycoplasma columbinium strain SF7 and comprises amino acid sequence as set forth in SEQ ID NO; 5 or an amino acid sequence having at least 40% similarity to SEQ ID NO: 5 after optimal alignment,
  • the protein or a derivative thereof is from Mycoplasma fermanta strain JER and comprises amino acid sequence as set forth in SEQ ID NO: 6 or an amino acid sequence having at least 40% similarity to SEQ ID NO:6 after optimal alignment,
  • the protein or a derivative thereof is from Mycoplasma pulmonis and comprises amino acid sequence as set forth in SEQ ID ' NO: 7 or an amino acid sequence having at least 40% similarity to SEQ ID NO: 7 after optimal alignment,
  • the protein or a derivative thereof is from Mycoplasma hyopneumomae and comprises amino acid sequence as set forth in SEQ ID NO:8 or an amino acid sequence having at least 40% similarity to SEQ ID NO:8 after optimal alignment.
  • the protein or a derivative thereof is from Mycophsm hyopneumomae and comprises amino acid sequence as set forth in SEQ ID NO: 16 or an. amino acid sequence having at least 40% similarity to SEQ ID NO: 16 after optimal alignment.
  • the protei or a derivative thereof is from Mycoplasma hyopneumomae and comprises amino acid sequence as set forth in SEQ ID NO: 1 7 or an amino acid sequence having at least 40% similarity to SEQ ID NO: 17 after optimal alignment.
  • the protein or a derivative thereof is from Mycoplasma hyopneumomae and comprises amino acid sequence as set forth in SEQ ID NO: 18 or an amino acid sequence having at least 40% similarity to SEQ ID NO: 18 after optimal alignment.
  • the protein or a derivative thereof is from Mycoplasma hyopneumwii e and comprises amino acid sequence as set forth in SEQ ID NO: 19 or an amino acid sequence having at least 40% similarity to SEQ ID NO: 19 after optimal alignment,
  • the protein or a derivative thereof is from Mycoplasm hyopneimtonia and comprises amino acid sequence as set forth in SEQ ID NO: 20 or an amino acid sequence having at least 40% similarity to SEQ ID NO: 20 after optimal alignment,
  • the protein or a derivative thereof is from Mycoplasma hyopneumomae and comprises amino acid sequence as set forth in SEQ ID NO:21 or an amino acid sequence having at least 40% similarity to SEQ ID NG:21 afte optimal alignment
  • the present specification teaches an isolated protein comprising an amino acid sequence selected from the list consisting of SEQ ID NOs: l throug 8 or 15 through 21.
  • Enabled herein is an isolated protein comprising an amino acid sequence set forth in SEQ ID NO :3.
  • Enabled herein is an isolated protein comprising an amino acid sequence set forth in SEQ ID NO :6.
  • Enabled herein is an isolated protein comprising an amino acid sequence set forth in SEQ ID NO: 1.7.
  • the present specification teaches the use of a protein or a derivative thereof having the amino acid sequence set forth in SEQ ID NQs: 1 through 8 or 15 through 21 or an amino acid sequence with at least 40% similarity to any of SEQ ID Os: 1 through 8 or 15 through 21 in the manufacture of a diagnostic agent to screen for current or prior exposure of a subject to a species of Mycoplasma.
  • the protein or derivative may also be used to screen for potential bovine respiratory disease, keratoconjunctivitis, mastitis, meningitis, otitis media and genital tract disease.
  • the present specification contemplates a method fo detecting current or prior exposure of a ruminant subject to a species of Mycoplasma, the method comprising contacting a antibody containing sample from the subject with an immobilized protein or derivative thereof comprising the amino acid sequence set forth in SEQ ID NO: l or an amino acid sequence having at least 40% similarity to SEQ ID NO: l after optimal alignment or an antibody -binding fragment of the protein, for a time and under conditions sufficient for an antibody specific for the protein, if present in the sample, to bind to the protein and then detecting for the presence of bound antibody wherein the presence of bound antibody is indicative of current or prior exposure to Mycoplasma spp.
  • the present specification further enables a method for detecting current or prior exposure of a ruminant subject to a species of Mycoplasma, the method comprising contacti g an antibody containing sample from the subject -with an immobilized protein or derivative thereof comprising the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence having at least 40% similarity to SEQ ID NO:2 after optimal alignment or an antibody-binding fragment of the protein, for a time and under conditions sufficient for an antibody specific for the protein, if present in the sample, to bind to the protein and then detecting for the presence of bound antibody wherein the presence of bound antibody is i dicative of current or prior exposure to Mycopktsma Spp.
  • the present specification further enables a method for detecting current or prior exposure of a ruminant subject to a species of Mycoplasma, the method comprising contacting an antibody containing sample from the subject with an immobilized protein or derivative thereof comprising the amino acid sequence set forth in. SEQ ID NO: 15 or an amino acid sequence having at least 40% similarity to SEQ ID NO:- 15 after optimal- alignment .or an antibody-binding fragment of the protein, for a time and under conditions sufficient for an antibody specific for the protein, if present in the sample, to bind to the protein and then detecting for the presence of bound antibody wherein the presence of bound antibody is indicative of current or prior exposure to Mycoplasma spp,
  • the protein comprises an amino acid sequence selected from the listing consisting of SEQ ID NOs2: through 8 or 15 through 21 or is an antibody- binding fragment thereof. [00841 In an embodiment, the protein comprises the amino acid sequence set forth in SEQ ID NO:2 of an amino acid sequence having at least 40% similarity to SEQ ID NO:2 after optima! alignment or is an antibody-binding fragment thereof.
  • the protein comprises the amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence having at. least 40% similarity to SEQ ID NO: 15 after optimal alignment or is an antibody-binding fragment thereof.
  • the protein comprises the amino acid sequence set forth in SEQ ID NO: 2 or is an antibody-binding fragment thereof.
  • the protein comprises the amino acid sequence set forth in SEQ ID NO: 15 oris an antibody -binding fragment thereof
  • the protein comprises the amino acid sequence set forth in SEQ ID NO; 16 through 21 or is an: antibody-binding fragment thereof.
  • Reference to a "ruminant subject” includes a cow, sheep, goat, giraffe, yak, camel, llama, antelope and macropod.
  • the species of Mycoplasma is M. bovis and the ruminant subject is a cow.
  • a “derivative” of the protein includes a fragment of the protein such as antibody-binding fragment. Fragments include MilA-AB, MilA-CD, Mil A- A, MilA-B, MilA-C, MilA-D; ilA-cd and MilA-ab.
  • a “derivative” also includes a protein having a single or multiple amino acid substitution, deletio and/or addition to SEQ ID NO: 1 or any one of SEQ ID NOs:2 through 8 or SEQ ID NOs; l5 through 21.
  • a ''derivative'' further includes the incorporation of an unnatural amino acid or chemical alteration of an amino acid side chain.
  • side chain modifications contemplated herein include modifications of amino groups such as by reductive aikylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with metliylacetimidate; acy!ation. with acetic anhydride; carbamoylation of amino groups with cyanate; tnnitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene suiphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyri doxy! ati on of lysine with pyridoxal-5-phosphate fol lowed by reduction with NaBHU.
  • modifications of amino groups such as by reductive aikylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with metliylacetimidate; acy!ation. with acetic anhydride; carbamoylation of amino groups with cyanate; tnnitrobenzylation of
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as :2 s 3-butariedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O- acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide,
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteie acid; formation of a mixed disulphides with other thiol compounds; reaction with nialeimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuripheriylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N- bromosuecmimide or aikylatio of the indole ring with 2-hydroxy-5-nitrohenzyl bromide or sulphonyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by aikylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include., but are not limited to, use of norleucine, 4-amino butyric acid, 4- amino-3-hydroxy-5-phenylpenianoie acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglyciiie, ornithine, sarcosine, 4-aniino-3-hydroxy-6-methylheptanoic acid, 2-t enyl alanine and/or D-isomers of amino acids,
  • Table 3 A list of unnatural amino acids, contemplated herein is shown in Table 3.
  • peptides can be Gonformationally constrained by, for example, incorporation of C e and N «-m ethyl amino acids, introduction of double bonds between C a and C atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the or C terminus.
  • any immunoassay may be used to target the MilA protein or its antibody- binding fragment or its derivative form.
  • Immunoassays are in effect binding assays. Examples of immunoassays include the various types of enzyme linked immunosorbent assays (ELS A), radioimmunoassays (RIA), Western blotting, dot blotting and FACS analyses.
  • ELS A enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • Western blotting Western blotting
  • dot blotting dot blotting and FACS analyses.
  • jOlOJ J In an. embodiment, an. EL-ISA is carried out using immobilized MilA protein. its honiolog, its derivative or an antibody-binding fragment (herein referred to as he "antigen"). In an embodiment, is immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate,
  • a sample putatively comprising antibodies to the naturally occurring Mil A protein is then added to the well s. After binding and washing to remove n on -specifically bound immune complexes, the antibody bound complex may be detected. Detection is generally achieved by the addition of a second antibody that is known to bind to the antibody and is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of the second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • the antibody-anti en complex is detected by a labeled antiimmunoglobulin antibody having an enzyme, chemiiuminescence, radioisotope, impedance or optical signal generating moiety attached thereto.
  • the anti -immunoglobulin activity is an an ti-IgG antibody.
  • the present specification further enables antibodies to Mil A or its homolog or derivative.
  • the antibodies may be monoclonal or polyclonal antibodies.
  • the antibodies are useful for screening for MilA protein or its homolo or derivative and for purifying or capturing MIA protein or its homolog or derivative.
  • antibody is meant a protein of the immunoglobulin family that is capable of combining, interacting or otherwise associating with an antigen (i.e. Mil protein or its homolog or derivative).
  • An antibody is, therefore, an antigen-binding molecule.
  • An “antibody” is an example of a immunointeractive molecule and includes a polyclonal or monoclonal antibody. Particularly useful immunointeractive molecules are monoclonal antibodies.
  • antigen is used herein in its broadest sense to refer to a MilA substance, or its honralogs or derivatives that are capable of reacting in and/or inducing an immune response.
  • antigen-binding molecule any molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins (e.g. polyclonal or monoclonal antibodies), immunoglobulin fragments and non- immufioglohulin derived protein frameworks that exhibit antigen-binding activity,
  • antigenic determinant or “epitope” is meant that part of an antigenic molecule against which a particular immune response is directed and includes an antibody-binding fragment of a MilA antibody. Typically, in an animal, antigens present several or even many antigenic determinants simultaneously.
  • Antibodies described herein may be from any source.
  • animal and avian sources and hosts include humans, primates, livestock animals (e.g. sheep, cows, horses, pigs, donkeys), laboratory test animals (e.g. mice, rabbits, guinea pigs, hamsters), companion animals (e.g. dogs, cats), poultry bird (e.g. chickens, ducks, geese, turkeys) and game birds (e.g. pheasants),
  • Immunization and subsequent production of monoclonal antibodies can be carried out using standard protocols as for example described by Kohler and Milstein (Kohler et ai (1975), Nature 256:495-499 and Kohler et at (1976), Eur. J. Immunol 6(7):5 11-519, Coligan et al. (19 1 -1997) Current Protocols in Immunology, or Toyama et ai. (1987), Monockma! Antibody, Experiment Manual, published by Kodansha Scientific.
  • an animal is immunized with an antigen-containing (i.e. MilA or a fragment or homolog thereof) by standard methods to produce antibody-producing cells, particularly antibody-producing somatic cells (e.g. B lymphocytes). These cells can then be removed from the immunized animal for immortalization.
  • the antigen may need to first be associated with a carrier.
  • carrier any substance of typically high molecular weight to which a non- or poorly immunogenic substance (e.g. a hapten) Is naturally or artificially linked to enhance its immunogenicity.
  • a non- or poorly immunogenic substance e.g. a hapten
  • Immortalization of antibody-producing cells may be carried out using methods, which are well-known in the art.
  • the immortalization may be achieved by the transfbrmation method using Epstein-Barr virus (EB V) (Kozbor et al (1986), Methods in EnzymoJogy 727: 140).
  • EB V Epstein-Barr virus
  • antibody-producing cells are immortalized using the cell fusion method (described in [Coligan et al. ⁇ " 1991-1997) supra]), which is widely employed tor the production of monoclonal antibodies.
  • somatic antibody- producing cells with the potential to produce antibodies, particularly B cells are fused with a myeloma cell line.
  • somatic cells may be derived from the lymph nodes, spleens and peripheral blood of primed animals, preferably rodent animals such as mice and rats.
  • rodent animals such as mice and rats.
  • murine spleen cells are used. It would be possible, however, to use rat, rabbit, sheep or goat cells, or cells from other animal species instead.
  • the second reaso ses from the inherent ability of lymphocytic tumor cells to produce their own antibodies.
  • myeloma cell lines incapable of producing endogenous light or heavy immunoglobulin chains are used.
  • a third reason for selection of these cell lines is for their suitability and efficiency for fusion.
  • Many myeloma cell lines may be used for the production of fused cell hybrids, including, e.g. P3X63-Ag8, P3X63-AG8.653, P3/NSl-Ag4-l (NS-1), Sp2/0-Agl4 and S 1 4/5.XXO.BU. 1.
  • the P3X63-Ag8 and NS-i cell lines have been described by Kohier and Milstein (Kohler et al. 1976 supra).
  • Shuiman et al. (1978) Nature 276:269-270, developed the Sp2/Q-Agl4 myeloma line.
  • the Sl 4/5.XXO.Bu.1 line was reported by Trowbridge (1982), J. Exp. Pied 148(1):22Q-2X1.
  • Methods for generating hybrids of antibody-producing splee or lymph node cells and m eloma cells usually involve mixing somatic cells with myeloma cells in a 10: ! proportion (although the proportion may vary from about 20: 1 to about 1 : 1), respectively, in the presence of an agent or agents (chemical, viral or electrical) that promotes the fusion of cell membranes. Fusion methods have been described ( ohler et al. (1975) supra, Kohler et al. (.1.976) supra, Gefter e i al. (1977), Somatic Cell Genet £.231.-236 and Volk et al. (1982) supra). The fusion-promoting agents used by those investigators were Sendai virus and polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • fusion procedures produce viable hybrids at very low frequency (e.g. when spleens are used as a source of somatic cells, only one hybrid is obtained for roughly every 1x10 " spleen cells), it is preferable to have a means of selecting the fused cell hybrids from the remaining unfused cells, particularly the unfused myeloma cells.
  • a means of detecting the desired antibody-producing hybridomas among other resulting fused cell hybrids is also necessary.
  • the selection of fused cell hybrids is accomplished by brieflyig the cell s in media that support the growth of hybridomas but prevent the growth of the unfused myeloma cells, which normally would go on dividing indefinitely.
  • The- somatic cells used in the fusion do not maintain long-term viability in in vitro culture and hence do not pose a problem.
  • myeloma cells lacking hvpoxanthine phosphoribosyl transferase HPRT -negative
  • HPRT hypoxanthine/aminopterin/thymidine
  • myeloma cells with different genetic deficiencies (drug sensitivities, etc) that can be selected against in media supporting the growth of genotypically competent hybrids is also possible.
  • each cell line may be propagated in either of two standard ways.
  • a suspension of the ybridoraa cells can be injected into a histocompatible animal. The injected animal will then develop tumors that secrete th specific monoclonal antibody produced b tire fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can be tapped to provide monoclonal antibodies in high concentration.
  • the individual cell lines may be propagated in vitro in laboratory culture vessels.
  • the culture medium containing high concentrations of a single specific monoclonal antibody can be harvested, by decantatioii, filtration or eentrifugation, and subsequently purified.
  • the cell lines are tested for their specificity to detect the antigen of interest by any suitable immunodetection means.
  • cell lines can be aliquoted into a number of wells and incubated and the supernatant from each well is analyzed by enzyme- linked immunosorbent assay (ELISA), indirect fluorescent antibody technique, or the like.
  • ELISA enzyme- linked immunosorbent assay
  • the cell iine(s) producing a monoclonal antibody capable of recognizing the target antigen but which does not recognize non-target epitopes are identified and then directly cultured in vitro or injected into a histocompatible animal to form tumors and to produce, collect and purify tire required antibodies.
  • the present specification is further directed to a compositio comprising a protein comprising an amino acid sequence set forth in SEQ ID NO: 1 or a derivative thereof or a protein having at lest 40% sequence similarity to SEQ ID NO: 1. in the manufacture of a medicament for the treatment or prophylaxis of infection by Mycoplasma species.
  • composition comprising a protein comprising an amino acid sequence set forth in SEQ 3D NQ:2 or a derivative thereof or a protein having at lest 40% sequence similarity to SEQ ID NO:2 in the manufacture of a medicament for the treatment or prophylaxis of infection by Mycoplasma species.
  • composition comprising a protein comprising an amino acid sequence set forth in SEQ ID NO: 15 or a derivative thereof or a protein having at lest 40% sequence similarity to SEQ ID NO: 15 in the manufacture of a medicament for the treatment or prophylaxis of infection by Kfycapl sm species,
  • the protein comprises the amino acid sequence set forth by SEQ ID NO:2 or an amino acid sequence having at least 40% similarity thereto or is a derivative of the protein.
  • the protein comprises the amino acid sequence set forth by SEQ ID NO: 15 or an amino acid sequence having at least 40% similarity thereto or is a derivative of the protein,
  • the protein comprises the amino acid sequence set forth by SEQ ID NO: 3 or an amino acid sequence having at least 40% similarity thereto or is a derivative of the protein.
  • the ⁇ protein comprises the amino acid sequence set forth by SEQ ID NO:4 or an amino acid sequence having at least 40% similarity thereto or is a derivative of the protein.
  • the protein comprises the amino acid sequence set forth by SEQ ID NO: 5 or an amino acid sequence having at least 40% similarity thereto or is a derivative of the protein. - 5J -
  • the protein comprises the amino acid sequence set forth by SEQ ID NO:6 or an amnio acid sequence having at least 40% similarity thereto or is a derivative of the protein.
  • the protein comprises the amino acid sequence set forth by SEQ ID NQ:7 or an amine- acid sequence having at least 40% similarity thereto or is a derivati ve of the protein.
  • the protein comprises the amino acid sequence set forth by SEQ ID NO: 8 or an amino acid sequence having at least 40% similarity thereto or is a deri ative of the protein.
  • the protein compri ses the amino acid sequence set forth by SEQ ID NO: 16 or an amino acid sequence having at least 40% similarity thereto or is a derivative of the protein.
  • the protein comprises the amino acid sequence set forth by SEQ ID NO: 17 or a amino acid sequence having at least 40% similarity thereto or is a derivative of the protein.
  • the protein comprises the amino acid sequence set forth by SEQ ID NO: 18 or an amino acid sequence having at least 40% similarity thereto or is a derivative of the protein,
  • the protein comprises the amino acid sequence set forth by SEQ ID NO: 19 or a amino acid sequence having at least 40% similarity thereto or is a derivative of the protein.
  • the protein comprises the amino acid sequence set forth by SEQ ID NO:20 or an amino acid sequence having at least 40% similarity thereto or is a derivative of the protein.
  • the protein comprises the amino acid sequence set forth by SEQ ID NO:21 or an amino acid sequence having at least 40% similarity thereto or is a derivative of the protein.
  • the protein is formulated, with a pharmaceutical carrier, excipient or diluent, which is non toxic to a ruminant subject,
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous).
  • any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavouring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches,, sugars, microcrystalMne cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques.
  • compositions disclosed herein suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension i an aqueous liquid, a non-aqueous liquid, an oil-in- water emulsio or a water-in-oi I liquid emulsion.
  • Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients.
  • compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
  • a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.
  • compositions may be administered orally, parenterally (including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques), by inhalation spray, or reetally, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, excipient and diluents.
  • the composition may also further comprise an adjuvant.
  • compositions When administered by nasal aerosol or inhalation, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
  • the protein may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
  • the injectable solutions or suspensions may be tonnulated according to known art, using suitable non-toxic, parenteraily-acceptable diluents or solvents, such as mannitol, 1 ,3-butanediol, water. Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • compositions When rectally administered in the form of suppositories, these compositions may be prepared by mixing the protein with a suitable non-irritating exctptent, such as cocoa butter, synthetic glycexide esters or polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the protein.
  • a suitable non-irritating exctptent such as cocoa butter, synthetic glycexide esters or polyethylene glycols, which are solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the protein.
  • the effective dosage of the protein employed in therapy may vary depending on the particular compound employed, the mode of administration, the conditio being treated and the severity of the condition being treated.
  • the dosage regimen utilizing the proteins of the present disclosure is selected in accordance with a variety of factors including type, species, age, weight, se and medical condition of the ruminant subject; the severity of the Mycoplasma infection to be treated; the route of administration; the renal and hepatic function of the subject; and the particular peptide employed.
  • a veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug requited to prevent, counter or arrest the progress of the condition.
  • Optimal precision in achieving concentratio of protei within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the protein's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of the protein.
  • Another aspect enabled herein is a method for controlling infection by a species of Mycoplasma in a subject, the method comprising administering to the subject an antibody-inducing effective amount of a protei or derivati ve thereof comprising the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 40% similarity to SEQ ID NO: I after optimal alignment for a time and under conditions sufficient to generate antibodies to the protein.
  • Another aspect enabled herein is a method for controlling infection by a species of Mycoplasma in a subject, the method comprising administering to the subject an antibody-inducing effective amount of a protei or derivative thereof comprising the amino acid sequence set forth in SEQ ID ' NO: or an amino acid sequence having at least 40% similarity to SEQ ID NO:2 after optimal alignment for a time and under conditions sufficient to generate antibodies to the protein.
  • Another aspect enabled herein is a method for controlling infection by a species of Mycoplasma in a subject, the method comprising administering to the subject an antibody-inducing effective amount of a protein or derivative thereof comprising the amino acid sequence set forth In SEQ ID NO: 15 or an amino acid sequence having at least 40% similarity to SEQ ID NO: 15 after optimal alignment for a time and under conditions sufficient to generate antibodies to the- protein,
  • the protein comprises the amino acid sequence set forth in SEQ ID NO:2 or a protein having at least 40% similarity to SEQ ID NO:2 or is a derivative thereof.
  • the protein comprises the amino acid sequence set forth in SEQ ID NO: 15 or a protein having at least 40% similarity to SEQ ID NO: 15 or is a derivative thereof.
  • the protein comprises the amino acid sequence set forth in SEQ ID NO:3 or a protein having at least 40% similarity to SEQ ID ' NO: 3 or is derivative thereof
  • the protein comprises the amino acid sequence set forth in SEQ ID NO:4 or a protein having at least 40% similarity to SEQ ID NO: 4 or is a derivative thereof
  • the protein comprises the amino acid sequence set forth in SEQ ID NO 5 or a protein having at least 40% similarity to SEQ ID NO 5 or is a derivative thereof
  • the protein comprises the amino acid sequence set forth in SEQ ID ⁇ 6 or a protein having at least 40% similarity to SEQ ID NO:6 or is a derivative thereof [0154] In an embodiment the protein comprises the amino acid sequence set forth in SEQ D NO:7 or a protein having at least 40% similarity to SEQ ID NQ:7 or is a derivative thereof.
  • the protein comprises the amino acid sequence set forth in SEQ ID NQ:8 or a protein having at least 40% similarity to SEQ ID NO; 8 or is a derivative thereof.
  • the protein comprises the amino acid sequence set fort in SEQ ID NO: 16 or a protein having at least 40% similarity to SEQ ID NO: 16 or is a deri ative thereof.
  • the protein comprises the amino acid sequence set forth in SEQ ID NO: 17 or a protein having at least 40% similarity to SEQ ID NO: 17 or is a derivative thereof.
  • the protein comprises the amino acid sequence set forth in SEQ ID NO: IS or a protein having at least 40% similarity to SEQ ID NO: 18 or is a derivative thereof.
  • the protein comprises the amino acid sequence set forth in SEQ ID NO: 1 or a protein having at least 40% similarity to SEQ ID NO: 19 or is a derivative thereof.
  • the protein comprises the amino acid sequence set forth in SEQ ID NO:20 or a protein having at least 40% similarity to SEQ ID NG:20 or is a derivative thereof.
  • the pnstera comprises the amino acid sequence set forth in SEQ ED NO:2I or a protein having at least 40% similarity to SEQ ID NO:21 or is a derivative thereof.
  • These aspects include conditions caused or exacerbated by Mycoplasma infection such as bovine respiratory disease, keratoconjunctivitis, mastitis, meningitis, otitis medi and genital tract disease.
  • the formulation of protein and its subsequent administration is within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several day s to several months, or until a cure is effected or a diminution of the disease state is achieved. Optim l dosing schedules can be calculated from measurements of drug accumulation in the subject. Persons or ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on. EC5 0 S found to be effective in in vitro and in vivo animal models.
  • dosage is from 0.01 ⁇ g to l.OOg per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or eve once every 2 to 20 years.
  • Persons of ordinary skill in the art can easily estimate repetition rats for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.
  • a ruminant subject is administered, an antibody to MilA or its homolog or derivative. This is regarded as passive immunotherapy. Antibodies to MilA or its homolog or derivative are generated as previously described herein.
  • Mycoplasma hovis wild-type strains 3683, 149040, PG45 and the temperature sensitive (is) mutant strains A, B and C were used.
  • M, bovis wild-type Strains (1/500 dilutions) were incubated for 17 h at 37 ° C in MB medium, and 1 /100 dilutions of the ts mutants were incubated for 24 h at 33 C C in MB medium.
  • pGEX-4T-l plasmid ( Figure 12) was used for cloning different regions of MilA and E. call JM109 cells were used to express recombinant GST-MilA proteins.
  • Experiment 2 Thirty five, one-month-old Friesian-cross calves were grouped according, to their weight and randomly grouped into 2 groups. Group 1 , the uninfected group, consisted of 5 calves exposed to an aerosol of mycoplasma culture medium, and Group 2 consisted of 30 cal ves exposed to an aerosol of M bovis strain 3683. Infection and sampling were performed as described for Experiment 1. [0.169] Experiment 3 - Group 3 cattle totaled 31 and were provided with vaccine A. SDS-PAGE and Western blots analysis
  • the total proteins were separated i 10% w/v or 12% w/v polyacryl amide separating gels by electrophoresis at 180 V for 1 h in the BioRad mini-vertical PAGE system containing Laemmli 's buffer (0.1 % w/v SDS, 0.025 M Tris, 0.19 M glycine). Broad range prestained (Fermentas) or unstained protein markers (New England Biolabs, USA), or biotinylaied protein markers (Cell Signaling Technology, USA) were used as molecular mass standards. The gels were stained with Coomassie brilliant blue -250 (BioRad, USA),
  • PVDF polyvinyls dene difluoride
  • the membrane was then washed three times (5 min each) with PBS-T [phosphate buffered saline (140 mM NaCl, 2,7 mM KC1, 10 mM Na 2 H 2 P04, mM HjPO*) containing 0, 1% v/v Tween 20 (Asia Pacific Specialty Chemicals, Australia)].
  • PBS-T phosphate buffered saline (140 mM NaCl, 2,7 mM KC1, 10 mM Na 2 H 2 P04, mM HjPO*) containing 0, 1% v/v Tween 20 (Asia Pacific Specialty Chemicals, Australia)].
  • the membrane was then probed with a pool of M bovh ⁇ -specific calf sera, diluted to 1/100 in 1% w/v skim milk in. PBS-T, and incubated for 1 h at room temperature with gentle rocking.
  • anti-M bovis rabbit antibody diluted to 1/20,000 in.
  • HRP horseradish peroxidase
  • Mycoplasma bovis cells from a 25 ml culture were pelleted by eentrifugatio at 16,000 g for 20 min at 4°C in a Sorvall . R.C-5B centrifuge (Sorvall, USA). The cell pellet was washed three times with PBS and resuspended in 0.25 ml PBS using a. 25 gauge needle and syringe. The cells were lysed with a 0.25 ml volume of 1% v/v Triton X-114 (Sigma, USA) and the mixture incubated on ice for 60 min, with mixing every 15 min. The ceil suspension was centrifuged at 13,000 ⁇ g for 30 min at 4 C.
  • the hydrophobic and hydrophiiic fractions were precipitated using the methanol/chloroform method.
  • To 1 volume of protein suspension 4 volumes of ice-cold methanol, 1 volume of ice-cold chloroform and 3 volumes of distilled water were added, the solutions vigorously mixed and then centrifuged for 1 min at 16,000 % g. The supernatant was discarded and the protein pellet resuspended i 4 volumes of methanol, vortexed and centnfuged for 5 min at 16,000 g at T, The supernatant was removed, the pellet dried under a vacuum and then dissolved in 8 M ur a. The proteins were then analyzed by SDS-PAGE or two dimensional electrophoresis (2 -DE).
  • sample preparation for two dimensional gel electrophoresis (2-DE) was carried out as described by Gorg (2004).
  • a .20 ml overnight, culture of M. bovi was pelleted by eentrifugation at 1.6,000 x g for 20 min at 4°C and the cell pellet washed three times with PBS, and resuspended using a 25 gauge needle i 0.5 ml of sample preparation solution [8 M urea, 4% w/v CHAPS (GE Healthcare, USA), 2% y/v IPG buffer pH 3 -1 1 (GE Healthcare, USA) and 40 mM DL-dithiothreitol (DTT) (Sigma- Aldrich.)].
  • the strip holder was placed on an Etlari TPGphorll isoelectric focusing system (Am ersharo -Bioscience) and the gel was re-swe) ' .led for 12 h at 30 V, then at 60 V for 6 h, and the isoelectric focusing performed for 5.5 h, at 200 V for 1 h, at 500 V for 1 h, at 1000 V for 1 h, at a gradient of 1000 V to 8000 V for 1 h, and at 8000 V for 1.5 h.
  • Etlari TPGphorll isoelectric focusing system Am ersharo -Bioscience
  • the strip was removed from the stri holder and equilibrated in SDS equilibration buffer (6 M urea, 75 mM Tris-HCi [pH 8.8], 87% v/v glycerol, 2% w/v SDS, 0.002% w/v bromophenoi blue with 10 mg . DTT/ml) for 15 min, followed by another 15 min with 25 mg jodoacetamide/ml (Am ersham, USA) substituted for the DTT.
  • the IEF strip gel was placed on top of a 12,5% w/v polyaerylamide SDS- PAGE gel arid sealed with agarose sealing solution (0.5% w/v agarose, 0.002% w/v bromophenoi blue in Laemmli's buffer) and the proteins separated according to their mass by electrophoresis. Once electrophoresis was completed the proteins were either transferred onto a PVDF membrane or stained with either colloidal Coomassie blue or silver (Plusone Silver Staining Kit-Protein, GE Healthcare, USA) according to the m an ufacturer ' s i nstru cti ons .
  • a 6 ⁇ b region of the M A gene was codon-optimized for expression in E. colt and the TG A tryptophan codons altered to TGG to confirm with E, coli codon usage.
  • the synthetic gene sequence was designed to contain a number of restriction endoimclease cleavage sites to aid manipulation in pGEX-4T-.l (BamHJL, Sail, XhoJ and Bgffi) [ Figure 1].
  • the modified DNA sequences were then manufactured by Genscript (USA) and cloned in pUC57.
  • the gene sequence optimized for expression in E. coli is set forth in SEQ ID Nos: 13 and 22.
  • the MilA-AB (SEQ ID NO: 13) and milA-CD (SEQ ID N0.22) fragments were excised from pUC57 using i1 ⁇ 43 ⁇ 4wHI, Bgll and 6 / ⁇ , purified by agarose gel electrophoresis and extraction with a QIAEX II kit (Qiagen, Germany) according to the manufacturer's instructions, and ligated in to pGEX-4T-l (GE Healthcare) that had been digested with Ba Kl and Sail.
  • the MilA-h, MlA- , M lA-C and MilA-D DNA fragments were excised from pGEXr4T-l -MilA -AB or pGEX-4T-l -.1 ⁇ 23 ⁇ 4-CD with appropriate restriction endoriucleases ( Figure I), purified, and ligated to similarly digested pGEX-4T-l ,
  • PGR was performed using 2 ng of pGEX-4T-l-M3 ⁇ 44-AB or pGEX-4T- i -M -CD DNA as the template and 0.4 ⁇ . ⁇ of the appropriate primers ( Figure I) in a reaction volume of 50 ⁇ containing 2 raM MgSQ , 100 ⁇ of each deoxynucleoside triphosphate and 1,5 U of Platinum Taq therm o- polyrnerase (Life Technologies).
  • Cycling conditions included an initial denaturation step at 94 °C for 4 rain, followed by 35 cycles of 94°C for I min, 55°C for i rain and 68°C for 2 min. Th 1.8 kb products were excised, purified and ligated into pGEX-4T- 1.
  • the ligated plasmids were used to transform E. coti JM109 cells by eleetrophoration (Noormohammadi et at. (1999) Microbiology 145: 2087-2094).
  • the transform ants were selected on LB agar containing ampicillin (50 g ml) and selected clones were incubated for 3 h at 30°C in the presence of 1 mM isopropyl -D-l-thiogalactopyranoside (IPTG) [Invitrogen], Expression of GST fusion proteins from the recombinant plasmids was assessed by SDS-PAGE and Western blotting of whole ceil lysates. Fusion protein GST- MilA-ab was purified by affinity chromatography using glutathione-Sepharose 4B beads (GE Healthcare) and elution with free glutathione (Crabb et ai. (1995) Arch. Virol. 7 ⁇ :245-258).
  • the vector pGEX-4T-l ( Figure 12) was used to transform E. colt JM109 cells by electroporation, according to the manufacturer's instructions (Gene Pulser, Bio Rad). The transformants were selected on LB agar containing ampicillin (50 ⁇ ⁇ 1.), and a single transformant was selected and grown overnight in LB broth supplemented with ampicillin (50 pg ml). The plasmid DNA was purified from the E, eoli cells using the Qiagen Plasmid Midi Kit (Qiagen) according to the manufacturer's instructions.
  • the pUC57-milA-AB and pUC57 -mi IA CD plasmids were digested with appropriate restriction endomicl eases (RE), BamHL, Bg and Sail, and pGEX-4T-1 was digested with BcmiHl and Sail according to the manufacturer's instructions (Ne England BioLahs).
  • the digested DNA was separated in a 1% w/v agarose gel and the required DNA bands (3 kb for the m/M-AB and milA-CU fragments and 5 kb for pGEX-4T-l ) were excised and the DNA extracted using the QIAEX II kit (Qiagen, Germany) according to the manufacturer's instructions.
  • the atnount of DNA was quantified by measuring the absorbance at 260 ran using a NanoDrop spectrophotometer (Biolab, Australia).
  • the milA- AB and milA-CD DNA fragments were ligated to similarly digested pGEX ⁇ 4T ⁇ l with T4 DNA ligase (Promega) in ligation buffer (Promega) at 4°C overnight
  • T4 DNA ligase Promega
  • ligation buffer Promega
  • Transfomiants were selected on LB agar containing ampiciUin (50 ⁇ %/ ⁇ ) and resistant transformants screened by PGR to confirm insertio of the mili-AB or milA-CD DN fragment in the pGEX-4T-l vector.
  • Those clones containing a 3 kb product as confirmed by PCR were selected and cultured overnight in LB broth supplemented with 50 g ampiciilin ml.
  • the plasmid DNA was purified using the Wizard Plus SV Mimpreps kit (Promega), digested ' with BarnHl and Sail and screened for the presence of the 3 kb insert and the 4. kb pGEX-4T-l vector by electrophoresis in a 0,8% w v agarose gel.
  • the clones with the expected RE digestion pattern were selected for expression.
  • a single transf rmant was used to set up an overnight culture in 5 ml LB broth containing 100 ⁇ g ampicillin/ml. The following day a 1/100 dilution of the overnight culture was used to inoculate 3 ml LB broth containing 100 ⁇ ampicillin/ml and this culture incubated at 37°C with shaking until it reached an OD ⁇ 3 ⁇ 4xi of 0,6-0.8 (around 2,5 h). A 1 ml sample of the culture was centrifuged at 1.6,000 x g for 5 mi and the cells resuspended in 100 ⁇ of PBS and stored at -20 ' C to be used as an uninduced sample.
  • JPTCi isopropyi ⁇ -D- l-thiogalactopyranoside
  • PCR was performed with 2 ng of pGEX-4T-l -miIA-AB plasmid DNA as the template with primers abF-ifcswH ' I and abR->$3 ⁇ 4r/I in a reaction volume of 50 ⁇ containing 2 mM MgS0 4 , 100 ⁇ of each deoxynucleoside triphosphate, 0,4 ⁇ V! of each primer and 1.5 U of Platinum Taq thermo-polymerase (Life Technologies). Cycling conditions included an initial denaturation step at 94°C for 4 min, followed by 35 cycles of 94°C for 1 mm, 5 for 1 min and 68"C for 2 min. The final cycle included an elongation step at 68"C for 5 min.
  • PCR products were resolved by electrophoresis in a 1% w/v agarose gel containing SyberSafe (Trade Mark) DNA gel stain (Molecular Probes) at 80 V for 1.5 ft and visualized under UV illumination using the ChemiDoc imaging system (BioRad).
  • SyberSafe Trade Mark DNA gel stain (Molecular Probes)
  • the 1 ,8 kb product was excised and the DNA extracted and digested with BamB md Sail as.
  • the digested DNA was purified using the Wizard Plus SV Mini rep kit (Promega) according, to the manufacturer's instructions.
  • a similar PG was performed to produce the 1.84 kb cd DNA fragment using the cdF-5 «wHI and cdK-Sail primers (Table 13), and the product digested and purified as above.
  • a single colony was used to inoculate 5 ml of LB broth containing 100 ttg ampieillin/ml and this culture incubated overnight at 37°C.
  • the 5 ml overnight culture was inoculated into 500 ml LB containing 100 pg ampicillin/ml and incubated at 37T with shaking until it reached an OD 6( JO of 0.6-0.8 (around 2,5 h),
  • a 1 ml sample of the culture was centrifuged at 16,000 % g for 5 min, resuspended in 100 ⁇ of PBS and stored at -20°C for use as the uninduced sample.
  • the cell suspension was sonicated 10 times for 10 sec each on ice and 1% v/v Triton X-100 (Plusone-Pharrnacia Biotech) added and the mixture vortexed and incubated for 30 min on ice. The mixture was then centrifuged at 6,500 x g for 1 min at 4°C and the pellet and supernatant were stored separately.
  • glutathione affinity column 1 ,33 ml of glutathione-sepharose 4B beads (GE Healthcare) were centrifuged at 500 ⁇ g for 5 min and the supernatant removed.
  • the beads were washed with PBS, centrifuged as above and the pellet of beads carefully placed in a Poly-Prep chromatography column (Bio ad) and left to settle at 4°C for 10 min.
  • the column was equilibrated by passing 5 ml of ice cold PBS through it.
  • the cell supernatant was filtered through a 0.45 pm nitrocellulose filter (Miliipore) and then placed on the column and allowed to pass through.
  • the column was then washed 3 times with 10 ml ice cold PBS and 10 ml of elution buffer (50 mM Tris-HCl [pH 8], 10 mM reduced glutathione [GE Healthcare]) was added to the column and left on it overnight at 4 0.
  • elution buffer 50 mM Tris-HCl [pH 8], 10 mM reduced glutathione [GE Healthcare]
  • Plasmid DNA of clones expressing the Mil A-AB, MilA-CD, Mil A- A, Mil A-B, MilA-C, MilA-D, Mil A-ab and MilA-ed proteins was extracted using the Wizard Plus SV Miiiipreps kit (Promega). Appfoximately 600 ng of plasmid DNA of each eonstract was used as the template in 20 ⁇ sequencing reactions containing the BDT sequencing terminator mix, BDT dilution buffer and 5 ⁇ of the primer (pGEXFwd or pGEXRev), The reaction was incubated at 96X fo 1 min, followed by 30 cycles of 96X for 10 s, 5 OX for 5 s and 60 X for 4 min.
  • reaction products were purified by adding 125 niM EDTA, 2.5 M sodium acetate and 100% v/v ethanol to a total volume of 74.4 ⁇ , and the mixture vortexed, incubated on ice for 1.5 min and centrifuged for 20 min at 16,000 x g at RT. The supernatant wa carefully removed and the pellet was washed in 70% v/v ethanol, centrifuged as above for 5 min, the supernatant discarded and the pellet dried by incubation in a heating block at 95 for 1 min.
  • the sequencing products were analysed by capillar)' sequencing and the sequences aligned to the predicted sequence using Geneious Pro 5.1.6.
  • the ELISA method used was adapted from Duffy et al (1999) J. Clin. Microbial 37; 1024-1029. Each well contained 1 ,2 ⁇ 3 ⁇ 4 of GST-Mil A-ab and blocking was performed with 5% v/v sheep serum in phosphate buffer saline. Dilutions of positive control, negative control and test sera were made in 2,5% v/v sheep serum in PBS -T (PBS containing 0.05% v/v Tween 20) by preparing two fold dilutions of the positive control calf serum, pool starting from 1/75, to 1/9600, as well as a single dilution of the negative control calf serum pool (1/300) and of test sera ( 1 /300).
  • HRP-eonjugated sheep anti-bovine IgG heavy and light chain antibody (Bethyl) was diluted at 1/2000 in 2.5% sheep serum in PBS-T and used as the conjugate. After the final wash, 10 ⁇ of 0.3 g/L 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) [ABTS Peroxidase Substrate, KPL] was added to each well and the plate incubated for 7 min at room temperature. The reaction was stopped by addition of 100 ⁇ of 1% w/v SDS to each well, and the absorbance of each well measured at 405 nm using a Hybrid Multimode microplate reader (BioTe ).
  • Test results from both experiments were pooled to calculate the sensitivity and specificity of the IgG ELISA.
  • the sensitivity was calculated separately for each time point and the sensitivity on day 24 after infection was considered to be the overall sensitivity of the IgG ELISA.
  • the specificity was calculated using one randomly selected sample from each of the uninfected calves (Group 1 -uninfected, Group 2-before infection, Group 3- vaccine A),
  • MHA Mycoplasma immunogenic lipase A
  • Mass spectrometry c analysis identified the coding sequence as that of MBO VPG45_710, which is predicted to be a 302.9 kDa membrane protein in . hi. bovis PG45 (accession number 313678759). Only the amino terminal 60% of the coding sequence was identified, with peptides derived from the remaining 40% not detected, explaining the lower molecular weight estimated for the protein from SD.S-polyaciylami.de gels.
  • the coding sequences of milA were synthesized as two 3 kb fragments, mi A- AB and mfM-CD, with B /rMl and Sail cleavage sites located at the amino and carboxyl terminal ends, respectively.
  • the two 3 kb fragments were able to be reduced in size by inclusion of Bgttl and Xhol.
  • PG and RE digestion screening also confirmed that the plasmids pGEX ⁇ 4T ⁇ 1 ⁇ //?/Z4 ⁇ AB, pGEX T-l.-»» 4-Cp, pGEX-4T-l-ra?M-A, pGEX-4T-.l ⁇ »iW-B, pGEX-4T-l-m/M-C and pGEX-4T-l-i»/M-D., which were cloned in E. eott JM109 cells, contained the expected inserts.
  • the predicted molecular weight (M ) of the recombinant proteins GST-MilA- AB, GST-MiiA-CD, GST-yil A-A, GST-MilA-B, GST-MilA-C and GST-MilA-D were 135,5 kDa, 137 kDa, 82.8 kDa, 79 kDa, 72 kDa and 73,3 kDa, respectively.
  • the actual MWs .of the recombinant proteins are summarized.
  • the GST-MiiA-CD, GST-MilA-B, GST-MilA-C and GST-MilA-D recombinant proteins were highly expressed, but did not react with M, bovis-specifw calf sera.
  • the GST-MiiA-AB and GST-MilA ⁇ A proteins reacted weakly with ⁇ ⁇ 5-specific calf sera. All the GST fusion proteins bound anti- GST antibody strongly.
  • the recombinant proteins GST-MilA-ab and GST-Mil A-cd were predicted to have MWs of 92.9 kDa and 94.3 kDa, respectively.
  • MBGVPG45_710 (milA) is predicted to code for a 302 fcDa protein of 2700 aa in the M. bovis reference strain PG45. Further analysis revealed that it contained a membrane spanning region at its amino terminal end, with the remainder of the protein predicted be located extraeellularly, Homologs were found in M. bovis strain Hubei-1, M. agalaciiae strains 5632 and PG2, cohrmhinunt SF7 and M sumam strains JER and 64 (Table 4).
  • SGNHJrydrolase region at the amino terminal end (aa 1-375) that belonged to a family of GDSL-like lipases ( Figure 5).
  • the SGNHJhydrolase region is characterized by the conserved catalytic site amino acids Ser, Gly, Asp and His.
  • the active site catalytic triad, Ser-His-Asp is in block 1 (Ser 99) and block V (His 362 and Asp 360).
  • I block I the conserved amino acid sequence is GDSL rather than the GDSL motif of the GDSL family lipases, as found in M. hyo eumomae mhp677 ⁇ Schmidt, 2004 #1241 ⁇ .
  • Gly is conserved
  • i block MI the sequence AXND is conserved in both M bovis PG45 and M.
  • infected calves had high IgG titers by day 24 ( Figure 9 and Table 6).
  • calves were above the cutoff value of 47,4 at day 10, but only one remained above the cutoff on days 17 and 24.
  • infected group four calves were positive on day 17 and twenty eight were positive on day 24,
  • the sensitivity of the IgG ELISA was 0% and 14,3% on days 10 and 17, respectively.
  • the overall sensitivity of IgG ELISA was 92,8% and specificity was 98,7%,
  • the sensitivity of the IgG ELISA for wild-type and vaccinated calves at each time point is shown in Table 9, The overall sensitivity of the IgG ELISA was 91 % and specificity was 98.4 %.
  • a 1/50 test serum dilution was selected, together with a HRP-conjjugate dilution of 1/2000, as this offered the best discrimination between infected and uninfected animals.
  • the development time for the ABTS substrate was increased to 20 min and the dilution series of the primary antibody for the standard curve for the test plate was 1/5, 1/10, 1/20, 1/40, 3/80, 1/ 160, 1/320 and 1/640.
  • the mean titer for the uninfected calves in the IgM ELISA was 99, and the cut off value was calculated to be 287 (99 + 188), On day 0, three animals were positive, but all were considered negative one week later. At day 14 after vaccination, 2 animals in Group 3 were positive, and the number of positive animals increased to 3, 5, 9 and 11 at each time point, Only one animal in Group 2 was positive before challenge, but the number increased to 11 and 18 on days 17 nd 24 after challenge (Table 1 1 and Figure 1 1 ).
  • the sensitivity of the IgM ELISA for wild-type and vaccinated calves at each time point is shown in Table 12.
  • the overall sensitivity of the IgM ELISA was 47,5 % and specificity was 98.8 .
  • experiment 1 had 24 calves, which were allocated into three groups. Grou 1 consisted of 4 calves exposed to aerosol of mycoplasma culture medium, Group 2 consisted of 12 calves exposed to Mycoplasm hovis strain 3683, while Group 3 consisted of 8 calves exposed to M bovis temperature sensitive vaccine strain A on days I and 3 and challenged together with calves from Grou 2 with bovisstimn 3683 three weeks after vaccination. Aerosol exposure was carried out as described before(Wawegama et al. (2012) supra).
  • the cut-off value for the IgG ELISA was calculated by considering all the uninfected calves in both experiments, including all the sera from Group .1 , sera from day 0 to day 18 from Group 2 and sera from day 0 from Group 3. The sensitivity was calculated at day 42 and the specificity was calculated for a randomly selected time point. [0223] The closed herd was considered as negative for M boviv and descriptive stati stics were computed and cut off value was calculated,
  • the mean antibody titer for the daily herd was calculated as 36 ⁇ 14.13, therefore, the cut-off was calculated as 64.3 AU. According to the cut-off all the cows except 2 were seiO-negative for M hovis and the 2 positive cows had antibody units less than 1.5 times the cut-off. Specificity of the IgG ELIS A using the dairy herd sera was calculated as 96%.
  • the M doiw-specific IgG ELISA is able to detect M, bovis infection in calves as well as adult cattle and can be used as a sera-diagnostic assay to screen for carriers for M. bovis.
  • MilA is a 226 kDa protein identified from M. b ' ovis strain 3683 by Western blotting. MilA is immunoreactive based on &oi7,v-speeific calf sera. MilA was estimated to be 226 kDa, but the gene encoding it, ,MBOVPG45__0710 (MilA), which was identified by mass speetrometri analysis of tryptie peptides derived from it, was predicted to encode a 30 kDa protein, suggesting that MilA may be cleaved into 2 peptides, as is see with the ciliary adhesin of M hyopneumomae,
  • Vsps variable surface proteins
  • the Vsps may have diagnostic utility (Brank et al. (1999) Clin, diagn. 1Mb. Immunol. 6; 861-867), they are subject to phase variation and antigenic variation, which may result in variations in the antibody response against them in individual infected cattle and compromise the sensitivity of the assay Poumarat et al. (1999) supra; Buchenau et al (2010) Res. Vet. Set 59:223-229). Therefore, use of a protein such as Mil A that is not subject to phase or antigenic variation,, but is still highly immunoreactive, is proposed to result in a useful diagnostic tool.
  • Geometri c mean of anti- bovis IgG titers

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Abstract

La présente invention concerne un test sérologique pour Mycoplasma et le développement de compositions prophylactiques et thérapeutiques pour traiter des sujets ruminants infectés par ou potentiellement exposés à des espèces Mycoplasma.
PCT/AU2014/050231 2013-09-16 2014-09-16 Test sérologique Ceased WO2015035474A1 (fr)

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Cited By (5)

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Publication number Priority date Publication date Assignee Title
CN107118262A (zh) * 2016-02-24 2017-09-01 华中农业大学 一种牛支原体MbovP579蛋白及其应用
CN110256555A (zh) * 2019-06-04 2019-09-20 中国农业科学院兰州兽医研究所 一种丝状支原体簇单克隆抗体及其制备方法和应用
CN110746504A (zh) * 2019-11-06 2020-02-04 华中农业大学 抗牛支原体MbovP579蛋白的单克隆抗体及其应用
CN112979767A (zh) * 2021-02-08 2021-06-18 山东省滨州畜牧兽医研究院 检测牛支原体抗体的抗原组合物、试剂盒及其应用
WO2022216162A1 (fr) * 2021-04-09 2022-10-13 Pictor Limited Dosage immunologique multiplex pour la détection d'une infection par mycoplasma bovis

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US5788962A (en) * 1995-01-17 1998-08-04 The Curators Of The University Of Missouri DNA sequences coding for mycoplasma hyopneumoniae surface antigens, corresponding proteins and use in vaccines and diagnostic procedures
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WAWEGAMA, N. K. ET AL.: "Development of a recombinant protein-based enzyme-linked immunosorbent assay for diagnosis of Mycoplasma bovis infection in cattle", CLINICAL AND VACCINE IMMUNOLOGY, vol. 21, no. 2, 2014, pages 196 - 202 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107118262A (zh) * 2016-02-24 2017-09-01 华中农业大学 一种牛支原体MbovP579蛋白及其应用
CN110256555A (zh) * 2019-06-04 2019-09-20 中国农业科学院兰州兽医研究所 一种丝状支原体簇单克隆抗体及其制备方法和应用
CN110746504A (zh) * 2019-11-06 2020-02-04 华中农业大学 抗牛支原体MbovP579蛋白的单克隆抗体及其应用
CN110746504B (zh) * 2019-11-06 2021-01-15 华中农业大学 抗牛支原体MbovP579蛋白的单克隆抗体及其应用
CN112979767A (zh) * 2021-02-08 2021-06-18 山东省滨州畜牧兽医研究院 检测牛支原体抗体的抗原组合物、试剂盒及其应用
WO2022216162A1 (fr) * 2021-04-09 2022-10-13 Pictor Limited Dosage immunologique multiplex pour la détection d'une infection par mycoplasma bovis
EP4320438A4 (fr) * 2021-04-09 2025-02-26 Pictor Limited Dosage immunologique multiplex pour la détection d'une infection par mycoplasma bovis

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