WO2012010849A2 - Méthodes de diagnostic et de vaccins - Google Patents

Méthodes de diagnostic et de vaccins Download PDF

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Publication number
WO2012010849A2
WO2012010849A2 PCT/GB2011/001104 GB2011001104W WO2012010849A2 WO 2012010849 A2 WO2012010849 A2 WO 2012010849A2 GB 2011001104 W GB2011001104 W GB 2011001104W WO 2012010849 A2 WO2012010849 A2 WO 2012010849A2
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mycoplasma
mhf
polypeptide
infection
seq
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WO2012010849A3 (fr
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Emily Ngaire Barker
Iain Robertson Peters
Christopher Richard Helps
Séverine TASKER
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University of Bristol
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University of Bristol
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    • 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
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/30—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]

Definitions

  • the present invention relates to methods of diagnosis of haemoplasma infection, and vaccines for the prevention of the same.
  • the methods and vaccines relate to the cat.
  • haemoplasmas is the trivial name given to a group of erythrocyte parasitizing bacteria of the genus Mycoplasma within the Mollicutes class. They are most closely related to the pneumoniae group of mucosal mycoplasmas. Analysis of 16S ribosomal RNA gene sequences from haemoplasmas resulted in their recent reclassification from the Haemobartonella and Eperythrozoon genera. The haemoplasma group can be further divided into haemofelis and haemominutum clades based on 15S rRNA and Ribonuclease P ribosomal gene phytogeny.
  • Diagnosis of haemoplasma infection was originally based on clinical signs and visualisation of epierythrocytic bodies by light microscopic examination of blood smears. More recently PCR technology has superseded cytology for diagnosis due to its superior sensitivity and specificity. PCR assays have also allowed accurate determination of the prevalence of the various haemoplasma species within their respective hosts. Development of serological assays for the diagnosis of haemoplasmosis has been attempted in the past. While early attempts by one group were reported to have been promising (unpublished data), no further information has been submitted for publication (Turner and others 1986).
  • Haemoplasmas have been found to infect a wide variety of mammalian species, from squirrel monkeys to llamas (Hoelzle 2008). And while they appear to be relatively host specific, different haemoplasmas may exist within an individual host (Kenny and others 2004, Tasker and others 2003, Willi and others 2006a).
  • CMhm prevalence ranges from 8.5 to 46.6%, Mhf prevalence from 0.2 to 21.7%, and CMt prevalence from 0.5 to 26.1 % (Bauer and others 2008, Fujihara and others 2007, Gentilini and others 2009, Kamrani and others 2008, Peters and others 2008, Roura and others 2010, Tasker 2006a, Willi and others 2006a, Willi and others 2006b).
  • Prevalence of the canine haemoplasmas ranges are even greater with Mhc prevalence from 0.9 to 20.0% and CMhp prevalence from 0.3 to 33.4%, with occasional dual-haemoplasma infections (Barker and others 2008, Inokuma and others 2006, Kenny and others 2004, Roura and others 2010, Wengi and others 2008).
  • haemotropic mycoplasmas is found in Tasker (2010). Infections with haemoplasmas can range from being asymptomatic, through mild pyrexia, to life- threatening (and occasionally fatal) haemolytic anaemia even in immunocompetent individuals. Of the feline haemoplasmas Mhf appears to be most significant in terms of haemolysis (Tasker 2006b).
  • CMhm Cats infected with CMhm are rarely clinically unwell, although a mildly lowered haematocrit is not uncommon when infected cats are compared to controls (George and others 2002, Tasker and others 2004, Tasker and others 2009b). A mildly lowered haematocrit was also seen in cats infected with CMt in a recent experimental study (Tasker and others 2009b). However, cats naturally infected with CMt are frequently found to be concurrently infected with one or more of the other haemoplasmas.
  • haemoplasma infection has been shown to modify response to viral and malarial infections, as well as response to irradiation (Baker and others 1971 , Contamin and Michel 1999).
  • the potential role of haemoplasma infection in modifying the pathogenicity of other infections has yet to be fully elucidated in cats.
  • CMhm infection has been associated with fatal myeloproliferative disease in chronically FeLV infected cats (George and others 2002), whilst other studies examining the effect of chronic FIV infection on acute Mhf or CMhm infection found no significant haematological differences between the retroviral positive and negative groups (Tasker and others 2006a, b). Long-term co-morbidity studies of natural cat populations have been limited by the presence of chronic infections and the lack of assays to determine exposure to haemoplasmas in the absence of a positive PCR result.
  • dnaK DNA chaperone heat shock protein 70
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • EF-Tu elongation factor thermo-unstable
  • EF-Tu of Mycoplasma pneumoniae has been shown to mediate binding to host fibronectin (Balasubramanian and others 2009). Some of these proteins are also immunogenic and have been suggested as vaccine candidates in particular species, such as GAPDH in the respiratory pathogen Mycoplasma bovis (Perez-Casal and Prysliak 2007).
  • GAPDH in the respiratory pathogen Mycoplasma bovis
  • a 40KDa immunoreactive GAPDH homolog of Ms, MSG1 has been shown to be expressed at both surface and cytosolic locations, have GAPDH activity and appeared to have a role in erythrocyte adhesion (Hoelzle and others 2007c).
  • a 70KDa immunoreactive protein of Ms, HspA1 was determined to be a dnaK homolog; a finding that was supported by ATPase activity of the expressed recombinant HspA1 (Hoelzle and others 2007b).
  • Recombinant MSG1 has been trialled as a vaccine candidate in the pig; however despite induction of anti-MSG1 antibodies they were not protective against a high-dose infection challenge (Hoelzle and others 2009).
  • In an analysis of the immunoproteome of Mycoplasma mycoides subsp. mycoides which infects cattle a number of proteins were identified as being immunogenic, including gapA, dnaK, EF-Tu and phosphoglycerate kinase (pgk) (Jores and others 2009).
  • Ms proteins MSG1 and HspA1 have been generated and used in ELISA-based serological diagnostic assays for the detection of Ms infection in pigs and was found to be comparable to whole cell preparations (Hoelzle and others 2007a).
  • a fragment of Mhf dnaK has also been expressed and used in western blots to detect the presence of anti-dnaK antibodies in cats experimentally infected with Mhf, CMhm or CMt (Museux and others 2009).
  • EF-Ts Elongation Factor - Temperature sensitive
  • gapA glycosyl-phosphate dehydrogenase
  • pgk phosphoglycerate kinase
  • Mhf or CMhm are useful as immunogenic marker proteins or in a vaccine for cats.
  • GapA and pgk are both metabolic proteins found in the cytoplasm of bacteria playing an integral role in glycolytic metabolic pathways. Due to reductive evolution mycoplasmas have lost a significant number of genes, as a result some proteins have evolved to function in multiple roles within the cell. It is impossible to predict which of these metabolic proteins will be used in this way in individual species, more so whether they will play a role in the bacterial immunome.
  • a first aspect of the invention provides a polypeptide comprising the amino acid sequence as set out in SEQ ID No 14 (Mhf EF-Ts) or a variant or fragment thereof.
  • SEQ ID No 14 provides the native amino acid sequence of Mhf elongation factor temperature- sensitive (EF-Ts).
  • EF-Ts forms part of a complex with elongation factors Tu and G, which together facilitate the events of translation (i.e. protein synthesis, where the DNA sequence is 'read' and amino-acids strung together).
  • EF-Ts serves as the guanine nucleotide exchange factor for EF-Tu, catalyzing the release of GDP from EF- Tu, allowing a molecule of GTP to bind in its place, so permitting the entry of the next aminoacyl rRNA into the ribosome. This may be assayed by incorporation into an ELISA, probing a western blot with plasma/serum or via enzymatic assays.
  • a second aspect of the invention provides a polypeptide comprising the amino acid sequence as set out in SEQ ID No 10 (Mhf pgk) or SEQ ID No 26 (CMhm pgk) or a variant or fragment thereof.
  • SEQ ID No 10 provides the partial native amino-acid sequence of Mhf phosphoglycerate kinase
  • SEQ ID No 26 provides the native amino acid sequence of CMhm phosphoglycerate kinase.
  • the Mhf pgk fragment comprised the N-terminal 344 amino-acids of what is suspected to be a total of 400-415 amino-acids of the complete protein.
  • Pgk is an ubiquitous enzyme that catalyses the formation of ATP to ADP and vice versa acting as a transferase enzyme in the seventh step of glycolysis ⁇ This could be assayed by incorporation into an ELISA, probing a western blot with plasma/serum or via enzymatic assays.
  • a third aspect of the invention provides a polypeptide comprising the amino acid sequence as set out in SEQ ID No 6 (Mhf gapA) or SEQ ID No 22 (CMhm gapA) or a variant of fragment thereof.
  • SEQ ID No 6 provides the native amino acid sequence of Mhf glyceraldehyde-3-phosphate dehydrogenase
  • SEQ ID No 22 provides the native amino acid sequence of CMhm glyceraldehyde-3-phosphate dehydrogenase.
  • GapA catalyzes the sixth step of glycolysis converting glyceraldehyde 3-phosphate to D- glycerate 1 ,3-bisphosphate.
  • a fourth aspect of the invention provides a polypeptide comprising the amino acid sequence as set out in SEQ ID No 18 (CMhm dnaK) or a variant or fragment thereof.
  • SEQ ID No 18 provides the native amino acid sequence of CMhm DNA chaperone heat shock protein dnaK.
  • the expression of heat shock proteins is up regulated in response to cell stressors such as heat or other environmental changes. More specifically heat shock protein 70, aka. dnaK, functions as an intra-cellular chaperone for other proteins permitting correct protein folding and preventing protein aggregation during unfavourable conditions. It may also be present at lower levels as a house-keeping protein fulfilling similar functions. This could be assayed by incorporation into an ELISA, probing a western blot with plasma/serum or via enzymatic assays.
  • a fifth aspect of the invention provides a polypeptide comprising the amino acid sequence as set out in SEQ ID No 2 (Mhf dnaK) or a variant thereof, or a fragment thereof with the amino acid sequence MSKKETIIG (SEQ ID No 29) or a fragment thereof from the sequence from position 309 to position 602 of Figure 3 (sequence B).
  • SEQ ID No 2 provides the native amino acid sequence of Mhf DNA chaperone heat shock protein dnaK.
  • Figure 3 indicates the amino-acid alignment between SEQ ID No 2 and the fragment of Mhf dnaK described by Museux and others (2009).
  • Position 309 onwards starts with the sequence QILLVGG (SEQ ID No 30) and so on, and ends with the sequence KTEVDKTKS (SEQ ID No 31).
  • the polypeptide is substantially free of other polypeptides with which it is naturally associated in the mycoplasma cell.
  • the polypeptide is substantially pure.
  • the polypeptide may be in a composition where it accounts for at least 95% of the total polypeptide, such as at least 96%, 97%, 98%, 99% or 99.5% as judged by Coomassie blue staining of a preparation separated by SDS-PAGE.
  • variant of the polypeptide we include polypeptides in which the sequence homology is at least 80% or 85% or 90%, more preferably at least 91 % or 92% or 93% or 94% or 95% or 96% or 97% or 98% or 99% or 99.5%, the % homology being assessed using the BLASTp algorithm [http://blast.ncbi.nlm.nih.gov/Blast.cgi, BLASTp (protein-protein BLAST), NCBI non-redundant protein sequence database (Altschul and others 1990).
  • the variants include polypeptides in which 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 amino acids have been replaced with another amino acid or have been deleted.
  • amino acids have been added to the polypeptide, for example in order to aid purification.
  • Suitable tags to facilitate purification include poly-histidine additions.
  • the variant retains the enzymatic active of the native polypeptide as given.
  • the variant is cross-reactive with the native polypeptide.
  • the variant is preferably able to bkid antibodies that have been raised to the native polypeptide, which can be tested for, for example by using a suitable ELISA.
  • fragment of the polypeptide we include fragments of the polypeptide that constitute 20% of the length of the native polypeptide, typically at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 97%.
  • the fragment is at least 20 amino acids, such as at least 30, 40, 50, 60, 70, 80 amino acids in length.
  • the fragment is cross- reactive with the native polypeptide.
  • the fragment is preferably able to bind antibodies that have been raised to the native polypeptide, which can be tested for, for example by using a suitable ELISA.
  • the fragment is either MSKKETIIG (SEQ ID No 29) or is a fragment from position 309 to position 602 of Figure 3 (sequence B).
  • polypeptide when used, unless the context does not allow it, the term includes the variants and fragments as defined including, in particular, the variants and fragments that are cross-reactive with the native polypeptide.
  • polypeptides and variants and fragments are typically immunogenic in the cat, giving rise to an immune response that can be measured by detecting antibodies to the polypeptide in the serum of a cat that has been immunised with the polypeptide or variant or fragment, for example by using an ELISA.
  • a sixth aspect of the invention provides a polynucleotide which encodes a polypeptide of any of the first, second, third, fourth or fifth aspects of the invention.
  • the polynucleotide is typically DNA, but may be RNA.
  • Preferred polynucleotides of the invention are SEQ ID No 1 , SEQ ID No 3, SEQ ID No 5, SEQ ID No 7, SEQ ID No 9, SEQ ID No 1 1 , SEQ ID No 13, SEQ ID No 15, SEQ ID No 17, SEQ ID No 19, SEQ ID No 21 , SEQ ID No 23, SEQ ID No 25 and SEQ ID No 27.
  • Codon usage refers to the triplet nucleotides that encode a specific amino-acid residue in a polypeptide chain or the termination of translation (stop codons).
  • the 'opal' codon UGA encodes for the amino-acid residue tryptophan, whilst in the majority of bacteria (including the Escherichia coli) UGA is a stop codon.
  • site-directed mutagenesis may be employed to convert the UGA codons to UGG, which also encodes the inclusion of tryptophan but is not translated as a stop codon in either the standard or mycoplasma codon usage tables.
  • a seventh aspect of the invention provides an expression vector comprising the polynucleotide of the sixth aspect of the invention.
  • the expression vector when present in a suitable host cell, allows for the expression of the polypeptides of the invention.
  • the expression vector may be a bacterial or yeast or insect cell or mammalian cell expression vector. It is preferred if the expression vector is a bacterial expression vector, such as an E. coli expression vector.
  • Suitable expression vectors are known in the art, for example in Sambrook et al Molecular Cloning, a laboratory manual, 2 nd edition, Cold Spring Harbor Laboraory Press, Card Spring Harbor, NY. ChampionTM pET Directional TOPO® Expression Kits (Invitrogen) manual part no. 25-0400 describes the use of this expression vector
  • An eighth aspect of the invention provides a host cell comprising a polynucleotide or expression vector of the invention.
  • the host cell may be any suitable host cell such as a bacterial cell, a yeast cell, an insect cell or a mammalian cell. Bacterial cells are preferred. Suitable host cells are known in the art, for example in Sambrook ef al.
  • polypeptides of the invention suitably are obtained by culturing a host cell which expresses the polypeptide by recombinant means, and then extracting the polypeptide from the host cell or culture medium (if secreted).
  • the polypeptide may be purified using methods well known in the art, such as ion-exchange chromatography, size-exclusion chromatography, affinity chromatography and the like.
  • purification using Ni 2* -affinity chromatography may be used.
  • the polypeptides of the invention may be packaged and presented for use in medicine, in particular in veterinary medicine.
  • the polypeptide or polypeptides of the invention may be packaged and presented for use in medicine.
  • the polypeptide is prepared as a sterile formulation, with appropriate pharmaceutical excipients, if required.
  • the polypeptide is substantially pure as described above.
  • the polypeptides, either alone or in combination with other antigenic proteins are useful as vaccines.
  • the vaccine of the invention therefore includes one or more polypeptides of the invention, optionally with other antigenic proteins, such as other mycoplasma proteins, and further optionally with one or more adjuvants.
  • Suitable adjuvants are known in the art and include, for example, alum.
  • a ninth aspect of the invention provides a method of vaccinating a cat against haemotropic mycoplasma infection, the method comprising administering to the cat one or more polypeptides according to any of the first, second, third, fourth or fifth aspects of the invention, or a vaccine according to the sixth aspect of the invention.
  • polypeptide includes variants or fragments thereof that are cross-reactive with the native polypeptide, and which are able to give rise to an immune response against the native polypeptide.
  • the polypeptide or polypeptides are administered by sub-cutaneous or intradermal or intra-muscular injection or by intra-nasal administration.
  • the amount of polypeptide or polypeptide is sufficient to give rise to a protective immune response in the cat.
  • western-blotting or ELISA may be used to determine whether the animal is able to produce an immune response to the recombinant protein itself.
  • animals may be challenged with infective doses of the selected haemoplasma at various time points following vaccine or placebo administration, then monitored for haemoplasma infection (clinical signs, haematological parameters and blood haemoplasma copy numbers).
  • haemoplasma infection clinical signs, haematological parameters and blood haemoplasma copy numbers.
  • a booster immunization is given at a suitable period after the initial immunization. If more than one t/pe of polypeptide is administered, they may be administered together or sequentially.
  • cat we include any member of the cat (felidae) family, in particular the domestic cat or wild felids such as tiger or lion.
  • An tenth aspect of the invention provides a composition comprising one or more polypeptides according to any of the first, second, third, fourth, fifth aspects of the invention and optionally other antigens, and optionally an adjuvant, or a vaccine according to the sixth aspect of the invention, for vaccinating a cat against haemotropic mycoplasma infection.
  • the invention also includes the use of a composition comprising one or more polypeptides according to any of the first, second, third, fourth, fifth aspects of the invention, or a vaccine according to the sixth aspect of the invention, in the manufacture of a medicament for vaccinating a cat against haemotropic mycoplasma infection.
  • the haemotropic mycoplasma infection is Mhf or CMhm or CMt.
  • An eleventh aspect of the invention provides an antibody directed to a polypeptide according to any of the first, second, third, fourth or fifth aspect of the invention.
  • Antibodies may be prepared by methods well known in the art, for example as described in “Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and Applications", J G R Hurrell (CRC Press, 1982), by using an appropriate antigen such as a polypeptide of the invention or a variant or fragment thereof which is cross-reactive with the native polypeptide, or a mycoplasma EF-Ts polypeptide or a variant or fragment thereof which is cross-reactive with the native polypeptide.
  • the antibody may be a polyclonal antibody or a monoclonal antibody or a recombinant antibody.
  • antibody we include fragment of antibodies such as Fab and Fv fragments, and synthetic antibody fragments such as scFv and dAb fragments.
  • the antibody is a specific antibody, in the sense that it binds the particular polypeptide but does not bind substantially to other polypeptides from the mycoplasma from which is may be obtained.
  • the antibody of the invention may be packaged and presented for use in medicine. Typically the antibody is prepared as a sterile formulation, with appropriate pharmaceutical excipients, if required.
  • a twelfth aspect of the invention provides a method of passively immunising a cat against haemotropic mycoplasma infection, the method comprising administering to the cat an antibody according to the eleventh aspect of the invention.
  • a sufficient amount of antibody is administered so as to provide protection against subsequent infection with mycoplasma.
  • the animal would be challenged with infective doses of the selected haemoplasma at various time points, then monitored for haemoplasma infection (clinical signs, haematological parameters and blood haemoplasma copy numbers).
  • the invention also includes an antibody according to the eleventh aspect of the invention for passively immunising a cat against haemotropic mycoplasma infection.
  • the invention also includes the use of an antibody according to the eleventh aspect of the invention in the manufacture of a medicament for passively immunising a cat against haemotropic mycoplasma infection.
  • haemotropic mycoplasma cats that have been infected with haemotropic mycoplasma are likely to contain antibodies directed at immunogenic polypeptides of the infecting haemotropic mycoplasma. They may also contain some of the immunogenic haemotropic mycoplasma polypeptides themselves. If the cat is diagnosed with haemotropic mycoplasma infection, typically it will be treated using antibiotics such as tetracyclines or fluoroquinolones. Supportive care may be given, such as fluid therapy if dehydrated, blood transfusion, or haemoglobin carrier solutions if severely anaemic. Appetite stimulation or nutritional support may be given if the cat is anorexic.
  • a thirteenth aspect of the invention provides a method of detecting whether a cat has been exposed to haemotropic mycoplasma infection, the method comprising determining whether a suitable sample from the cat is reactive with a polypeptide according to any of the first, second, third, fourth or fifth aspects of the invention.
  • the sample is a fluid sample from the cat which is known to contain antibodies. Suitable samples include whole blood, plasma, serum, or saliva.
  • the method comprises detecting the presence of antibodies in the sample which are reactive towards the polypeptide. Suitable methods for determining the presence of antibodies are known in the art, for example ELISA methods (enzyme-linked immunosorbent assay) as is discussed in more detail below.
  • a fourteenth aspect of the invention provides a method of detecting whether a cat has been exposed to haemotropic mycoplasma infection, the method comprising determining whether a sample from the cat contains one or more polypeptides according to the first, second, third, fourth or fifth aspects of the invention.
  • Suitable samples include whole blood, plasma, serum, urine, tissues, faeces, or saliva.
  • Antibody-based methods useful for detecting polypeptides in a sample include immunoassays, such as the enzyme linked immunosorbent assay (ELISA), western-blot and the radioimmunoassay (RIA).
  • a polypeptide-reactive monoclonal antibody can be used both as an immunoadsorbent and as an enzyme-labeled probe to detect and quantify the polypeptide.
  • the amount of polypeptide present in the sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm.
  • Such an ELISA for detecting a tumour antigen is described in Lacobelli et al, Breast Cancer Research and Treatment 1 1: 19-30 (1988).
  • two distinct specific monoclonal antibodies can be used to detect polypeptide in a body fluid. In this assay, one of the antibodies is used as the immunoadsorbent and the other as the enzyme-labeled probe.
  • the above techniques may be conducted essentially as a "one-step” or “two-step” assay.
  • the "one-step” assay involves contacting a sample potentially containing the polypeptide with immobilized antibody and, without washing, contacting the mixture with the labeled antibody.
  • the "two-step” assay involves washing before contacting the mixture with the labeled antibody.
  • Other conventional methods may also be employed as suitable. It is usually desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed from the sample.
  • Suitable enzyme labels include, for example, those from the oxidase group, which catalyze the production of hydrogen peroxide by reacting with substrate.
  • Glucose oxidase is particularly preferred as it has good stability and its substrate (glucose) is readily available.
  • Activity of an oxidase label may be assayed by measuring the concentration of hydrogen peroxide formed by the enzyme-labeled antibody/substrate reaction.
  • radioisotopes such as iodine ( 125 l, 12 l), carbon ( 14 C), sulfur 35 S), tritium ( 3 H), indium ( 12 ln), and technetium ( 99 mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • CMhm gapA polypeptide is reactive against serum from feline post-CMhm infection but not reactive against serum from feline post-Mhf infection.
  • Differentiation of infecting species is important as Mhf is pathogenic, resulting in significant disease during acute infection, whilst CMhm less frequently results in disease, but is less readily cleared from the infected animal.
  • Animals infected with Mhf would most likely require antibiosis and supportive therapy (e.g. fluid therapy including blood transfusions, appetite stimulation) whilst those infected with CMhm rarely require therapy in the absence of concurrent disease unless attempted clearance of infection is desired.
  • CMhm dnaK polypeptide of the fourth aspect of the invention
  • Mhf dnaK polypeptide of the fifth aspect of the invention
  • the methods of the thirteenth and fourteenth aspects of the invention may be considered to be methods of diagnosing whether a cat is or has been infected with haemotropic mycoplasma.
  • the invention also includes an immunosorbent assay for detecting anti-mycoplasma protein antibodies in a sample, the assay comprising a solid phase coated with a polypeptide according to any of the first, second, third, fourth or fifth aspects of the invention wherein the anti-mycoplasma protein antibodies in a sample exposed to the solid phase will bind to the polypeptide, and a detectable label conjugate which will bind to the anti-mycoplasma protein antibodies bound to the solid phase.
  • the invention also includes a solid substrate with a polypeptide according to any of the first, second, third, fourth or fifth aspects of the invention attached thereto.
  • the solid phase or solid substrate is a microtitre well or plate.
  • the conjugate comprises an anti-cat antibody.
  • the conjugate comprises a detectable moiety such as an enzyme, for example horseradish peroxidise or alkaline phosphatase.
  • the immunosorbent assay also contains a substrate for the enzyme.
  • the assay comprises a solid phase coated with a suitable polypeptide (such as one of the polypeptides of the first or second or third or fourth or fifth aspect of the invention, or a mycoplasma EF-Ts polypeptide, wherein anti-mycoplasma antibodies in a sample exposed to the solid phase will bind to the polypeptide; and a detectable label conjugate which will bind to the anti-CRCV antibodies bound to the solid phase.
  • a suitable polypeptide such as one of the polypeptides of the first or second or third or fourth or fifth aspect of the invention, or a mycoplasma EF-Ts polypeptide, wherein anti-mycoplasma antibodies in a sample exposed to the solid phase will bind to the polypeptide; and a detectable label conjugate which will bind to the anti-CRCV antibodies bound to the solid phase.
  • an antigenic fragment of the polypeptide that coats the solid phase is of sufficient size to be bound by an anti-mycoplasma antibody.
  • the haemotropic mycoplasma polypeptide, or antigenic variant or fragment thereof, that coats the solid phase is at least 10 amino acids in length. More preferably, the haemotropic mycoplasma polypeptide, or antigenic variant or fragment thereof, is at least 20, or at least 30, or at least 40, or at least 50, or at least 100 amino acids in length.
  • the invention also includes a kit of parts which include the components of the immunosorbent assays.
  • the kit of parts may thus include a solid phase such as a microtitre plate, the appropriate haemotropic mycoplasma protein for coating the solid phase, a detectable label conjugate, such as an anti-cat antibody, which will bind to anti- mycoplasma antibodies bound to the solid phase. If the detectable label conjugate is an enzyme, the kit of parts may also include a substrate for the enzyme.
  • the kit may also include a positive control sample that contains an anti-mycoplasma antibody, such as those described with reference to the eleventh aspect of the invention, and a negative control sample.
  • protein is coated on microtitre plates overnight at 4°C to 37°C, depending on the stability of the antigen. Unbound protein is washed off with a wash buffer such as phosphate buffered saline or Tris buffered saline. Serum or other samples are incubated on the plate, typically at 37°C for between 1 and several hours. Unbound material is washed off, the plates are incubated with enzyme-labelled (e.g.
  • horseradish peroxidase or alkaline phosphatase antibody such as anti-host mammal (such as ant-feline) IgG or IgM for plasma/whole blood/serum samples, or anti-host mammal (such as anti-feline) IgA for saliva, for 1 to several hours at 37°C. Unbound antibody is washed off and plates are incubated with a substrate such as OPD for about 10 min where horseradish peroxidase was used, or p-nitrophenyl phosphate for about 75 min where alkaline phosphatase was used, and the optical density measured in a photometer.
  • a substrate such as OPD for about 10 min where horseradish peroxidase was used, or p-nitrophenyl phosphate for about 75 min where alkaline phosphatase was used, and the optical density measured in a photometer.
  • the polypeptide is a native haemotropic mycoplasma polypeptide.
  • FIG. 1 Western blot of RBC membrane antigen (R) and Mhf antigen (Hf) probed with plasma samples (1 :250) collected pre-infection (-7 and 0) and post-infection (15, 29, 43, 57, 71, 85, 99, 11 1 , 125, 139 and 153 DPI) from cat HF4.
  • the calculated molecular mass for the major bands (kDa) are shown.
  • the open triangle corresponds to the 71- 72kDa band 4.2 protein identified in both RBC and Mhf antigen preparations.
  • FIG. 2 A Comassie blue-stained PAGE gel (A) of RBC membrane antigen (R) and a Western blot (B) of a pooled plasma sample (P: days -7 and 0, all cats) and RBC membrane antigen (R) probed with the pooled plasma sample (1 :250).
  • the molecular mass (kDa) of each protein standard (S) is given on the left side.
  • the calculated molecular mass (kDa) of each identified band is given on the right side with the likely RBC membrane proteins corresponding to the bands (3, 24).
  • FIG 4 Two-dimensional electrophoresis of Mhf protein stained with Sypro Ruby. Immunogenic spots picked for mass-spectrometry indicated. Proteins were separated initially according to isoelectric point on a pH3-11 NL IPG strip (horizontal), then according to mass on a 8% poly-acrylamide gel (vertical; Precision Plus Protein standard on right hand side - major bands 75, 50, 25 kDa indicated).
  • FIG. 5 Mhf protein preparation separated by two-dimensional electrophoresis and western blotted with post-infection plasma from two Mhf infected cats. The pH orientation and three molecular mass markers are indicated. The three matched spots are indicated in white.
  • FIG. 7 Recombinant Mhf dnaK (rMhf dnaK) ELISA optical density results for plasma taken immediately prior to, and during, acute CMhm infection (HM2). Prior to infection no reactions towards rMhf dnaK were detected. At 29 days post-infection the cat demonstrated a weak reaction to rMhf dnaK, which decreased over the following 42 days.
  • Example 1 Antigen Specificity of the Humoral Immune Response to Mycoplasma haemofelis Infection
  • the aim of this study was to characterise the antigenic specificity of the humoral immune response made by cats infected with the feline haemoplasma, Mycoplasma haemofelis (Mhf).
  • a crude Mhf antigen preparation was prepared from red blood cells (RBCs) collected from a cat at a time of high bacteraemia. Plasma samples were collected from six cats pre- and post-experimental infection with Mhf, with regular sampling performed from 15 to 149 or 153 days post-infection (DPI). Pre-infection RBC membrane ghosts were prepared from these six cats, and used to identify erythrocyte proteins that may have contaminated the Mhf antigen preparation.
  • the Mhf antigen preparation comprised 11 protein bands.
  • the immunodominant bands on western blotting with infected cat plasma were of molecular mass 78, 68, 60, 48 and 38kDa.
  • Most cats (n 5) had plasma antibody that reacted with at least one band (always including 68kDa) at 15 DPI and all cats were seroreactive by 29 DPI.
  • the maximum number of antibody specificities from an individual animal was identified in plasma collected between 57 and 99 DPI. Contamination of the Mhf antigen preparation with RBC membrane proteins was observed.
  • the contaminating RBC proteins were of molecular mass 71-72kDa (consistent with band 4.2) and 261 and 238kDa (consistent with spectrin) and these were recognised by all plasma samples.
  • a range of Mhf antigens are recognised by cats infected experimentally with the organism. These represent possible targets for immunoassays, but care must be taken to prevent false positive results due to host protein contamination.
  • plasma samples were collected for 22 weeks following Mhf infection, to enable more extensive characterisation of proteins recognised by the immune system of infected cats and to attempt to determine whether autoantibodies specific for erythrocyte membrane antigens were induced by the infection.
  • infection of the cats was carried out by obtaining heparinised blood from barrier- maintained donor cats chronically infected with Mhf.
  • the blood type of the donor cats and all recipients was predetermined to be Type A (RapidVet-H blood typing cards, DMS Laboratories Inc, New Jersey, US).
  • Fresh blood was collected from donors and injected intravenously into the recipients.
  • These inocula comprised 2 ml of heparinised blood (infective dose HF14: 7.21 ⁇ 10 7 , others: 1.55 * 10 8 Mhf copies) given via pre-placed cephalic intravenous catheters within 5 min of collection from the donors. All procedures and experiments described were undertaken under a project license approved under the UK Animals (Scientific Procedures) Act 1986.
  • EDTA- anticoagulated blood samples were regularly collected from all cats and subjected to real-time PCR (qPCR) to confirm that Mhf infection was absent before inoculation, and present post-infection (Tasker and others 2009a; Tasker and others 2009b). Seven days before infection (day -7), on the day of infection (day 0), and approximately every 2 weeks thereafter, until day 149 (HF2) or 153 (HF1 , 4, 6, 8 and 12), an additional 1 ml volume of EDTA anticoagulated blood was collected from each cat, centrifuged (2200 * g, 3 min) and the plasma removed and stored at -20°C until use.
  • qPCR real-time PCR
  • a pooled plasma sample was made by mixing an equal volume of the pre-infection plasma samples collected during the study.
  • Plasma samples, from EDTA blood (the RBC were used for other purposes e.g. Coombs' test), were used in order to minimize the total volume of blood collected from the animals and to comply with the terms of the studies' project license.
  • RBCs were sedimented by centrifugation at 600g for 10 min, plasma and buffy coat were aspirated, and the RBCs were washed and centrifuged in an equal volume of phosphate-buffered saline (PBS; 0.15M, pH 7.4). The PBS and remaining buffy coat was aspirated and the wash repeated twice more. The final packed, washed RBC were incubated in PBS containing 0.15% Tween-20 and 3% (w/v) EDTA, and incubated for 30 min at ambient temperature, in a vertical shaker (speed 150rpm) to dislodge the Mhf.
  • PBS phosphate-buffered saline
  • Debris and erythrocytes were pelleted by centrifugation at 600g for 10 min and the resultant supernatant was centrifuged at 40,000g for 30 min at 4°C to pellet the Mhf. The supernatant was removed, the pellet resuspended in 1 ml PBS and centrifuged through 20% sodium diatrozoat meglumine and diatrozoat sodium (76% Urografin; Bayer pic, Newbury, Berkshire, UK) at 15,000rpm for 40min at 4°C, and the final pellet was resuspended in 1.0 ml PBS.
  • the resulting Mhf antigen preparation was depleted of any contaminating albumin or IgG using the ProteoExtract Albumin/lgG Removal Kit (Calbiochem, Merck Chemicals Ltd., Nottingham, UK) according to the manufacturer's instructions.
  • the flow-through and the wash from the columns were added to a 20K iCON concentrator (Pierce Biotechnology, Fisher Scientific UK Ltd., Loughborough, Leicestershire, UK) and centrifuged at 6,000g for 1 hour.
  • the protein concentration in the retained solution was measured using a Qubit (Invitrogen Ltd., Paisley, Scotland) before storage at -80°.
  • RBC membrane ghosts were prepared from 1 ml EDTA anticoagulated blood from the six plasma source cats (HF1 , 2, 4, 6, 8 and 12), prior to infection, using the methods described by Barker (Barker 1991 ). Briefly, RBCs from 1 ml EDTA anticoagulated blood were washed six times in 10 volumes of PBS with aspiration of the supernatant and buffy-coat between washes. RBCs were lysed by adding 10 volumes of ice-cold lysis buffer (20mM Tris, pH 7.6) and pelleted by centrifugation at 40,000g for 30min at 4°C. The supernatant and opaque buttons of residual leucocytes were removed, using a pipette, leaving the translucent RBC membranes.
  • the membranes were washed a minimum of four times in ice-cold lysis buffer until the supernatant was clear of haemoglobin.
  • the concentration of protein in the final solution was measured by Qubit and stored at -80°C prior to use. PAGE and Western Blot Analysis
  • PAGE Polyacrylamide gel electrophoresis
  • NuPAGE electrophoresis system Invitrogen Ltd., Paisley, UK
  • proteins were transferred to nitrocellulose membranes using the XCell mini-cell and Blot Module (Invitrogen, Paisley, UK) as per the manufacturer's instructions.
  • NuPAGE Novex 4-12% Bis-Tris precast gels, NuPAGE MOPS running buffer, NuPAGE LDS sample buffer (all Invitrogen Ltd) and Mhf or RBC membrane antigen (2Q ⁇ g per well) were used for the investigation of the humoral immune responses.
  • Protein transfer was performed using the NuPAGE transfer buffer (Invitrogen) and a pre-stained molecular weight standard (All Blue Precision Plus Protein Standard, Bio-Rad Laboratories Ltd., Hemel Hempstead, UK) was included on each gel to monitor transfer efficiency and to allow calculation of molecular mass,
  • the NuPAGE Large Protein Blotting Kit (Invitrogen Ltd.), which included 3-8% tris-acetate gels, the HiMark Pre-Stained High Molecular Weight Protein Standard and 0. 45 ⁇ nitrocellulose membranes, was used to further investigate antigenic proteins in the RBC membrane preparations, as this gave better separation of the high molecular weight proteins.
  • nitrocellulose membranes were blocked with 5% (w/v) non-fat milk in TBST (50 mM Tris, 150 mM NaCI, 0.05% Tween 20, pH-7.6) for 2 hours. Membranes were then cut in to strips, each including a lane of Mhf antigen and RBC membrane ghosts from the individual cat. The strips were probed overnight with 1 in 250 dilution of plasma (D-7, 0, 15, 29, 43, 57, 71 , 85, 99, 111 , 125, 139 and 149 or 153) in TBST + 5% non-fat milk.
  • D-7 0, 15, 29, 43, 57, 71 , 85, 99, 111 , 125, 139 and 149 or 153
  • a negative control omitting incubation with a plasma sample, was performed to control for reactivity between secondary antibody and the Mhf antigen and RBC membrane ghosts.
  • the antibody specific for the 71-72kDa protein identified in the RBC ghosts and the Mhf antigen preparations was present in plasma collected pre- (days -7 and 0) and post-infection, although the intensity of this band varied when plasma samples taken at different time points were compared for the same animal. This band was not considered to be of Mhf origin and was excluded from the analysis of the immune response to Mhf. None of the 12 pre-infection plasma samples contained antibody to any of the other 11 Mhf bands. No protein bands were identified when the secondary antibody alone was incubated with the antigen preparations (data not shown).
  • Mhf antigen preparations Contamination of Mhf antigen preparations with host blood-derived proteins is a recognized problem and such contaminants may originate from the cellular components (RBC and white blood cells) and the plasma.
  • blood was collected from an experimentally infected cat at the time of presumptive peak Mhf copy number (1 1 DPI), and before the onset of anaemia and Coombs' test positivity (Tasker and others 2009b). Blood collected after the onset of anaemia and Coombs' positivity was susceptible to severe haemolysis during the isolation procedure, resulting in haemoglobin and RBC membrane contamination of the Mhf preparations (unpublished observations).
  • a 71-72kDa protein band was detected in the Mhf and RBC membrane antigen preparations by all pre and post-infection plasma samples, but was not detected when the secondary antibody was used alone.
  • This protein band has molecular mass equivalent to the erythrocyte protein band 4.2 (Korsgren and others 1990; Steck and others 1974), and correlated with band 4.2 identified on a Commassie blue-stained PAGE gel of RBC membrane proteins. This band was not of a size that would be consistent with immunoglobulin light chains or ⁇ or ⁇ heavy chains identified in the blot of the pooled plasma sample.
  • the human anti-spectrin antibodies have been shown to cross-react with a protein migrating to an equivalent position as band 4.2 (Lutz and Wipf 1982). These physiological autoantibodies specific for erythrocyte proteins have a role in the removal of damaged and senescent RBC (reviewed by Kay 2005).
  • This broad protein band may therefore be composed of host-derived proteins. The size of this protein would be consistent with that of immunoglobulin light chain, but the band was not recognized by the secondary antibody alone. Further work is required to determine the identity of this band.
  • Mhf The major antigenic determinants of Mhf, to which infected cats respond serologically, were of molecular mass 78, 68, 60, 48 and 38kDa. All cats produced antibodies reactive with all of these bands by 57 DPI. A previous study found significant reactivity to Mhf determinants of molecular mass 150, 52 and 14 kDa by 30 DPI (Alleman and others 1999). Antibodies reactive with Mhf proteins of 53 and 54kDa were found in the plasma of cats in the present study, but the former specificity was only identified in one cat and the latter was seen in only four cats by 57 DPI, which is the time point closest to the last sample (60 DPI) analysed in the study by Alleman ef al.
  • mice infected with M. haemomuris develop serum antibodies to Mycoplasma determinants of molecular mass 118, 65, 53, 45, and 40 kDa at 42 DPI (Rikihisa and others 1997).
  • Pigs infected with Msu produce antibodies reactive with major MSu proteins of 70, 45 and 40kDa and minor proteins of 83, 73, 61 , 57, and 33kDa.
  • antibody specific for the major bands was present as early as 7 DPI and was well- established by 14 DPI (Hoeize and others 2006).
  • the variation in the molecular mass of the Mhf determinants detected in our study compared with those from previous studies may be due to the experimental methods used or the haemoplasma species investigated.
  • the major determinants described in the Msu study may correspond to Mhf determinants of 68, 48 and 38kDa described in the present study, but use of a different acrylamide gel concentration (4-12% gradient vs 10%) and buffer system in the two studies may have resulted in slightly different migration rates, affecting the calculation of molecular mass.
  • these differences may reflect variations between the Mycoplasma species as they reside in different clades within the haemoplasmas as determined by 16S rDNA and RNaseP phylogenetic analysis (Johansson and others 1999; Messick and others 2002; Neimark and others 2001 ; Peters and others 2008; Tasker and other 2003).
  • HspA1 70kDa protein
  • MSG1 40kDa protein
  • erythrocyte-bound antibody (as opposed to the circulating antibody investigated here) is specific for surface Mhf antigens or erythrocyte autoantigens and whether the antigenic specificity of antibody eluted from the erythrocytes of Mhf-infected cats differs from that of the circulating antibody pool.
  • Example 2 Identification of antigens specific for the humoral immune response to Mhf, CMhm and CMt infection
  • haemoplasma DNA Materials & Methods Sources of haemoplasma DNA. haemoplasma protein, host protein and pre- & post- immune plasma All procedures and experiments described were undertaken under a project license approved under the Animals in Scientific Procedures Act, 1986. Additional data from these experiments has been described in published reports (Peters and others 2010, Tasker and others 2009a, Tasker and others 2009b).
  • the high-speed pellet of haemoplasmas was subjected to differential gradient centrifugation, and subsequently concentrated using a 9 KDa molecular weight cut-off filter. A similar extraction procedure from uninfected erythrocyte membranes was also performed to obtain a negative control.
  • EDTA anti-coagulated blood was taken from barrier maintained SPF cats prior to and weekly thereafter following experimental infection with Mhf, CMhm or CMt.
  • Excess EDTA anti-coagulated blood from samples submitted to Langford Veterinary Services Diagnostic Laboratories (LVS-DL) for companion animal haemoplasma PCR were also collected. Blood was centrifuged and separated. Plasma was stored at -20°C until required. Partial sequencing of the haemoplasma genome
  • Haemoplasma genomic DNA was purified using Macherey-Nagel NucleoSpin® Blood kits, from the haemoplasma DNA rich supernatant. Extracted DNA was sheared by nebulisation into 2 to 5 Kbp fragments, as determined by gel electrophoresis. These fragments were ligated into pCR ® 4Blunt-TOPO ® and transformed into One Shot ® Chemically Competent Escherichia coli (TOPO ® Shotgun Subcloning Kit; Invitrogen). Purified plasmids from randomly selected clones were sequenced using dye-terminator chemistry with piasmid and custom primers.
  • Mhf organisms and un-infected erythrocyte membrane protein extracts were prepared for 2D SDS-PAGE using the Amersham 2D-clean up kit (GE Life Sciences). Proteins were initially separated according to isoelectric point on ImmobilineTM DryStrip pH 3-11 NL (GE Life Sciences), then according to mass (12.5% polyacrylamide gel). Gels were either western blotted onto a polyvinylidene difluoride (PVDF) membrane or stained using Sypro ® Ruby Protein Gel Stain (Invitrogen).
  • PVDF polyvinylidene difluoride
  • Membranes were blocked by gentle agitation for 5 hours in tris-buffered saline (25mM Tris, 150mM NaCI) with Tween-20 (0.1 % v/v) and 5% (w/v) non-fat milk (TBST-5M). Membranes were incubated overnight with test plasma (1 :500 dilution of plasma from each of two cats taken at different time point: 'pre-infection' - from 7 and 1 days pre-infection; 'post-infection' - from 139 and 153 days post-infection) in TBST-5M.
  • test plasma (1 :500 dilution of plasma from each of two cats taken at different time point: 'pre-infection' - from 7 and 1 days pre-infection; 'post-infection' - from 139 and 153 days post-infection
  • Washed membranes were incubated for 2 hours at room temperature in TBST-5M containing 1 :20,000 dilution of alkaline phosphatase conjugated goat anti-cat IgG (H+L) antibody.
  • Membranes were washed in Tris-buffered saline (25mM Tris, 150mM NaCI) with Tween-20 (0.1% v/v) (TBST) between steps (20 minutes; two wash changes; orbital shaker at 25rpm; room temperature). Following the final washes, visualisation was achieved using Lumi-Phos WBTM Chemiluminescent Substrate and exposure to Amersham Hyperfilm ECLTM.
  • the Mhf gapA, Mhf dnaK and CMhm gapA protein coding sequences were cloned into pET101/D-TOPO ® and expressed in BL21 StarTM (DE3) E. coli as fusion proteins with C- terminal His-tags (ChampionTM pET Directional TOPO ® Expression Kit, Invitrogen).
  • CMhm gapA coding sequence Prior to cloning the CMhm gapA coding sequence was subjected to site directed mutagenesis to convert opal stop codons (TGA) to tryptophan codons (TGG). His-tagged proteins were purified using the Ni-NTA Spin Kit (QIAgen).
  • Proteins were separated using one- dimensional gel electrophoresis (NuPAGE ® Novex ® Bis-Tris Gel System) and western blotted onto nitrocellulose. Product size was determined by comparison to a molecular weight ladder (Precision Plus ProteinTM Standards; Bio-Rad). Expressed protein bands were excised from the NuPAGE gels and subjected to trypsin-digestion and mass- spectrometry analysis to confirm their identity. Immunoblots of recombinant haemoplasma proteins
  • Wells were incubated with plasma (duplicates of 1:200, 1 :400 and 1 :800 dilutions in ⁇ /well PBST-10M) for 2 hours at room temperature under agitation then washed with PBST.
  • Wells were then incubated with either anti-cat IgG conjugated with alkaline phosphatase (heavy and light chains; Jackson ImmunoResearch) or anti-dog IgG conjugated with alkaline phosphatase (heavy and light chains; Sigma) at 1 :10,000 dilution in 100pl/well PBST-10M for 2 hours at room temperature under agitation and then washed with PBST.
  • Each plate contained wells to which (i) no plasma was added (triplicate), (ii) haemoplasma negative SPF feline plasma (1 :200, 1 :400 and 1 :800 dilutions) was added, (iii) duplicate two-fold serial dilutions of a strong positive feline plasma (1 :200 to 1 :409,600) was added.
  • a serum sample was deemed positive if its OD value was greater than the OP value of the 1 :51 ,200 dilution of the positive control (typically an OD of 0.100 at ⁇ 75min).
  • Anti-Mhf dnaK antibodies were detected using 1 D immunoblots in cats experimentally infected with Mhf, whilst cross-reactive antibodies were detected in cats experimentally infected with CMhm and CMt and in a dog naturally infected with Mhc. This suggests that an assay using rMhf dnaK could be used to detect exposure to haemotropic mycoplasmas in companion animals.
  • haemoplasma proteins pgk and EF-Ts
  • pgk and EF-Ts have been identified as immunogenic ie they are recognised with Mhf post-infection serum from cats, indicating suitability for use in a vaccine and as a marker of haemotropic mycoplasma infection.
  • Haemoplasma genomic DNA was purified using Macherey-Nagel NucleoSpin® Blood kits, from the haemoplasma DNA rich supernatant. Extracted DNA was sheared by nebulisation into 2 to 5 Kbp fragments, as determined by gel electrophoresis. These fragments were ligated into pCR ® 4Blunt-TOPO ® and transformed into One Shot ® Chemically Competent E. coli (TOPO ® Shotgun Subcloning Kit; Invitrogen). Purified plasmids from randomly selected clones were sequenced using dye-terminator chemistry with plasmid and custom primers.
  • BLASTn analysis of nucleotide sequences and BLASTp analysis of the corresponding amino-acid sequences was performed to identify the origin of the insert sequence (http://blast.ncbi.nlm.nih.gov/Blast.cqi).
  • Target specific primers were used in conjunction with plasmid primers to obtain contiguous sequence by PCR using Mhf and CMhm libraries as template.
  • VVAINDLTDP TLAHLLKYDTAHGPVRCYDISV EGDSIVLVNKCSGEKQSFKVISERDP ALPWKSL
  • VDCVLECTGRFTDKDAAMAHVEAGA KKWISAPAKGDLKTIVY VNHGTLTSSDQVISAASCTTNALAPWDALHKKYKIVSGF
  • MSG1 a surface- localised protein of Mycoplasma suis is involved in the adhesion to erythrocytes.

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Abstract

L'invention concerne des polypeptides immunogènes de Mycoplasma haemofelis (Mhf) et 'Candidatus Mycoplasma haemominutum' (CMhm), ainsi que leurs utilisations en tant que vaccins et marqueurs de l'infection chez le chat. Les polypeptides sont dnaK, gapA, pgk et EF-Ts.
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Non-Patent Citations (65)

* Cited by examiner, † Cited by third party
Title
ALLEMAN, A. R., M. G. PATE, J. W. HARVEY, J. M. GASKIN, A. F. BARBET.: "Western immunoblot analysis of the antigens of Haemobartonella felis with sera from experimentally infected cats", J CLIN MICROBIOL, vol. 37, 1999, pages 1474 - 9, XP002662570
ALTSCHUL, S. F., GISH, W., MILLER, W., MYERS, E. W., LIPMAN, D. J.: "Basic local alignment search tool", JOURNAL OF MOLECULAR BIOLOGY, vol. 215, 1990, pages 403 - 410, XP002949123, DOI: doi:10.1006/jmbi.1990.9999
ALVAREZ, R. A., BLAYLOCK, M. W., 6ASEMAN, J. B.: "Surface localized glyceraldehyde-3-phosphate dehydrogenase of Mycoplasma genitalium binds mucin", MOLECULAR MICROBIOLOGY, vol. 48, 2003, pages 1417 - 1425
BAKER, H. J., CASSELL, G. H., LINDSEY, J. R.: "Research complications due to Haemobartonella and Eperythrozoon infections in experimental animals", AMERICAN JOURNAL OF PATHOLOGY, vol. 64, 1971, pages 625 - 632
BALASUBRAMANIAN, S., KANNAN, T. R., HART, P. J., BASEMAN, J. B.: "Amino acid changes in elongation factor-Tu of Mycoplasma pneumoniae and Mycoplasma genitalium influence fibronectin binding", INFECTION AND IMMUNITY, 2009
BALLAS, S. K.: "Spectrin autoantibodies in normal human serum and in polyclonal blood grouping sera", BR J HAEMATOL, vol. 71, 1989, pages 137 - 9
BARKER, E. N., TASKER, S., DAY, M. J., WOOLLEY, K., BIRTLES, R. J., GEORGES, K., EZEOKOLI, C. D., CLEAVELAND, S., HELPS, C. R.: "Development and use of novel real-time PCR assays for canine haemotropic mycoplasmas", 18TH EUROPEAN COLLEGE OF VETERINARY INTERNAL MEDICINE - COMPANION ANIMALS CONGRESS, 2008, pages 198
BARKER, R. N., T. J. GRUFFYDD-JONES, C. R. STOKES, C. J. ELSON: "Identification of autoantigens in canine autoimmune haemolytic anaemia", CLIN EXP IMMUNOL, vol. 85, 1991, pages 33 - 40
BARKER, R. N.: "Electrophoretic Analysis of Erythrocyte Membrane Proteins and Glycoproteins from Different Species", COMP HAEMATOL INT, vol. 1, 1991, pages 155 - 160
BAUER, N., BALZER, H. J., THURE, S., MORITZ, A.: "Prevalence of feline haemotropic mycoplasmas in convenience samples of cats in Germany", JOURNAL OF FELINE MEDICINE AND SURGERY, vol. 10, 2008, pages 252 - 258, XP022690368, DOI: doi:10.1016/j.jfms.2007.12.004
BERENT, L. M., MESSICK, J. B., COOPER, S. K.: "Detection of Haemobartonella felis in cats with experimentally induced acute and chronic infections, using a polymerase chain reaction assay", AMERICAN JOURNAL OF VETERINARY RESEARCH, vol. 59, 1998, pages 1215 - 1220
CONTAMIN, H., MICHEL, J. C.: "Haemobartonellosis in squirrel monkeys (Saimiri sciureus): antagonism between Haemobartonella sp. and experimental Plasmodium falciparum malaria", EXPERIMENTAL PARASITOLOGY, vol. 91, 1999, pages 297 - 305
FUJIHARA, M., WATANABE, M., YAMADA, T., HARASAWA, R.: "Occurrence of 'Candidatus Mycoplasma turicensis' infection in domestic cats in Japan", JOURNAL OF VETERINARY MEDICAL SCIENCE, vol. 69, 2007, pages 1061 - 1063
GENTILINI, F., NOVACCO, M., TURBA, M. E., WILLI, B., BACCI, M. L., HOFMANN-LEHMANN, R.: "Use of combined conventional and real-time PCR to determine the epidemiology of feline haemoplasma infections in northern Italy", JOURNAL OF FELINE MEDICINE AND SURGERY, vol. 11, 2009, pages 277 - 285, XP025974124, DOI: doi:10.1016/j.jfms.2008.06.008
GEORGE, J. W., RIDEOUT, B. A., GRIFFEY, S. M., PEDERSEN, N. C.: "Effect of preexisting FeLV infection or FeLV and feline immunodeficiency virus coinfection on pathogenicity of the small variant of Haemobartonella felis in cats", AMERICAN JOURNAL OF VETERINARY RESEARCH, vol. 63, 2002, pages 1172 - 1178
H ZOLA: "Monoclonal Antibodies: A manual of techniques", 1988, CRC PRESS
HACKETT, T. B., JENSEN, W. A., LEHMAN, T. L., HOHENHAUS, A. E., CRAWFORD, P. C., GIGER, U., LAPPIN, M. R.: "Prevalence of DNA of Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum', Anaplasma phagocytophilum, and species of Bartonella, Neorickettsia, and Ehrlichia in cats used as blood donors in the United States", JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION, vol. 229, 2006, pages 700 - 705
HOELZLE, K., DOSER, S., RITZMANN, M., HEINRITZI, K., PALZER, A., ELICKER, S., KRAMER, M., FELDER, K. M., HOELZLE, L. E.: "Vaccination with the Mycoplasma suis recombinant adhesion protein MSG1 elicits a strong immune response but fails to induce protection in pigs", VACCINE, 2009
HOELZLE, K., GRIMM, J., RITZMANN, M., HEINRITZI, K., TORGERSON, P., HAMBURGER, A., WITTENBRINK, M. M., HOELZLE, L. E.: "Use of recombinant antigens to detect antibodies against Mycoplasma suis, with correlation of serological results to hematological findings", CLINICAL AND VACCINE IMMUNOLOGY, vol. 14, 2007, pages 1616 - 1622
HOELZLE, L. E., D. ADELT, K. HOELZLE, K. HEINRITZI, M. M. WITTENBRINK: "Development of a diagnostic PCR assay based on novel DNA sequences for the detection of Mycoplasma suis (Eperythrozoon suis) in porcine blood", VET M ICROBIOL, vol. 93, 2003, pages 185 - 96, XP002423344, DOI: doi:10.1016/S0378-1135(03)00040-3
HOELZLE, L. E., HOELZLE, K., HARDER, A., RITZMANN, M., AUPPERLE, H., SCHOON, H. A., HEINRITZI, K., WITTENBRINK, M. M.: "First identification and functional characterization of an immunogenic protein in unculturable haemotrophic Mycoplasmas (Mycoplasma suis HspA1", FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY, vol. 49, 2007, pages 215 - 223, XP009080119, DOI: doi:10.1111/j.1574-695X.2006.00184.x
HOELZLE, L. E., HOELZLE, K., HELBLING, M., AUPPERLE, H., SCHOON, H. A., RITZMANN, M., HEINRITZI, K., FELDER, K. M., WITTENBRINK, M: "MSG1, a surface-localised protein of Mycoplasma suis is involved in the adhesion to erythrocytes", MICROBES AND INFECTION, vol. 9, 2007, pages 466 - 474, XP005934792
HOELZLE, L. E., HOELZLE, K., RITZMANN, M., HEINRITZI, K., WITTENBRINK, M. M.: "Mycoplasma suis antigens recognized during humoral immune response in experimentally infected pigs", CLINICAL AND VACCINE IMMUNOLOGY, vol. 13, 2006, pages 116 - 122, XP002423343, DOI: doi:10.1128/CVI.13.1.116-122.2006
HOELZLE, L. E.: "Haemotrophic mycoplasmas: Recent advances in Mycoplasma suis", VETERINARY MICROBIOLOGY, vol. 130, 2008, pages 215 - 226, XP025425788, DOI: doi:10.1016/j.vetmic.2007.12.023
INOKUMA, H., OYAMADA, M., DAVOUST, B., BONI, M., DEREURE, J., BUCHETON, B., HAMMAD, A., WATANABE, M., ITAMOTO, K., OKUDA, M.: "Epidemiological survey of Ehrlichia canis and related species infection in dogs in eastern Sudan", ANNALS OF THE NEW YORK ACADEMY OF SCIENCES, vol. 1078, 2006, pages 461 - 463
J G R HURRELL: "Monoclonal Hybridoma Antibodies: Techniques and Applications", 1982, CRC PRESS
JOHANSSON, K. E., J. G. TULLY, G. BOLSKE, B. PETTERSSON: "Mycoplasma cavipharyngis and Mycoplasma fastidiosum, the closest relatives to Eperythrozoon spp. and Haemobartonella spp", FEMS MICROBIOL LETT, vol. 174, 1999, pages 321 - 6
JORES, J., MEENS, J., BUETTNER, F. F., LINZ, B., NAESSENS, J., GERLACH, G. F.: "Analysis of the immunoproteome of Mycoplasma mycoides subsp. mycoides small colony type reveals immunogenic homologues to other known virulence traits in related Mycoplasma species", VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY, vol. 131, 2009, pages 238 - 245, XP026600304, DOI: doi:10.1016/j.vetimm.2009.04.016
KAMRANI, A., PARREIRA, V. R., GREENWOOD, J., PRESCOTT, J. F.: "The prevalence of Bartonella, hemoplasma, and Rickettsia felis infections in domestic cats and in cat fleas in Ontario", CANADIAN JOURNAL OF VETERINARY RESEARCH, vol. 72, 2008, pages 411 - 419
KAY, M.: "Immunoregulation of cellular life span", ANN N Y ACAD SCI, vol. 1057, 2005, pages 85 - 111
KEMMING, G., MESSICK, J. B., MUELLER, W., ENDERS, G., MEISNER, F., MUENZING, S., KISCH-WEDEL, H., SCHROPP, A., WOJTCZYK, C., PACKE: "Can we continue research in splenectomized dogs? Mycoplasma haemocanis: Old problem - New insight", EUROPEAN SURGICAL RESEARCH, vol. 36, 2004, pages 198 - 205
KENNY, M. J., SHAW, S. E., BEUGNET, F., TASKER, S.: "Demonstration of two distinct hemotropic mycoplasmas in French dogs", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 42, 2004, pages 5397 - 5399
KORSGREN, C., J. LAWLER, S. LAMBERT, D. SPEICHER, C. M. COHEN: "Complete amino acid sequence and homologies of human erythrocyte membrane protein band 4.2", PROC NATL ACAD SCI U S A, vol. 87, 1990, pages 613 - 7
LACOBELLI ET AL., BREAST CANCER RESEARCH AND TREATMENT, vol. 11, 1988, pages 19 - 30
LESTER, S. J., HUME, J. B., PHIPPS, B.: "Haemobartonella canis infection following splenectomy and transfusion", CANADIAN VETERINARY JOURNAL, vol. 36, 1995, pages 444 - 445
LOBETTI, R. G., TASKER, S.: "Diagnosis of feline haemoplasma infection using a real-time PCR assay", JOURNAL OF THE SOUTH AFRICAN VETERINARY MEDICAL ASSOCIATION, vol. 75, 2004, pages 94 - 99
LUTZ, H. U., G. WIPF: "Naturally occurring autoantibodies to skeletal proteins from human red blood cells", J JMMUNO, vol. 128, 1982, pages 1695 - 9
MESSICK, J. B., P. G. WALKER, W. RAPHAEL, L. BERENT, X. SHI.: "Candidatus Mycoplasma haemodidelphidis' sp. nov., 'Candidatus Mycoplasma haemolamae' sp. nov. and Mycoplasma haemocanis comb. nov., haemotrophic parasites from a naturally infected opossum (Didelphis virginiana), alpaca (Lama pacos) and dog (Canis familiaris): phylogenetic and secondary structural r", INT J SYST EVOL MICROBIOL, vol. 52, 2002, pages 693 - 8
MUSEUX, K., BORETTI, F. S., WILLI, B., RIOND, B., HOELZLE, K., HOELZLE, L. E., WITTENBRINK, M. M., TASKER, S., WENGI, N., REUSCH,: "In vivo transmission studies of 'Candidatus Mycoplasma turicensis' in the domestic cat", VETERINARY RESEARCH, 2009, pages 40,45
NEIMARK, H., K. E. JOHANSSON, Y. RIKIHISA, J. G. TULLY: "Proposal to transfer some members of the genera Haemobartonella and Eperythrozoon to the genus Mycoplasma with descriptions of 'Candidatus Mycoplasma haemofelis', 'Candidatus Mycoplasma haemomuris', 'Candidatus Mycoplasma haemosuis' and 'Candidatus Mycoplasma wenyonii", INT J SYST EVOL MICROBIOL, vol. 51, 2001, pages 891 - 9
PEREZ-CASAL, J., PRYSLIAK, T.: "Detection of antibodies against the Mycoplasma bovis glyceraldehyde-3-phosphate dehydrogenase protein in beef cattle", MICROBIAL PATHOGENESIS, vol. 43, 2007, pages 189 - 197, XP022258348, DOI: doi:10.1016/j.micpath.2007.05.009
PETERS, . R., C. R. HELPS, L. MCAULIFFE, H. NEIMARK, M. R. LAPPIN, T. J. GRUFFYDD-JONES, M. J. DAY, L. E. HOELZLE, B. WILLI, M. ME: "RNaseP RNA Gene (rnpB) Phylogeny of Haemoplasmas and Other Mycoplasma Species", J CLIN MICROBIOL, 2008
PETERS, . R., HELPS, C. R., GRUFFYDD-JONES, T. J., DAY, M. J., TASKER, S.: "Antigen specificity of the humoral immune response to Mycoplasma haemofelis infection", JOURNAL OF CLINICAL MICROBIOLOGY, 2010
PETERS, . R., HELPS, C. R., WILLI, B., HOFMANN-LEHMANN, R., TASKER, S.: "The prevalence of three species of feline haemoplasmas in samples submitted to a diagnostics service as determined by three novel real-time duplex PCR assays", VETERINARY MICROBIOLOGY, vol. 126, 2008, pages 142 - 150, XP022351071, DOI: doi:10.1016/j.vetmic.2007.06.017
RIKIHISA, Y., M. KAWAHARA, B. WEN, G. KOCIBA, P. FUERST, F. KAWAMORI, C. SUTO, S. SHIBATA, M. FUTOHASHI: "Western immunoblot analysis of Haemobartonella muris and comparison of 16S rRNA gene sequences of H. muris, H. felis, and Eperythrozoon suis", J CLIN MICROBIOL, vol. 35, 1997, pages 823 - 9, XP002926817
ROURA, X., PETERS, . R., ALTET, L., TABAR, M. D., BARKER, E. N., PLANELLAS, M., HELPS, C. R., FRANCINO, 0., SHAW, S. E., TASKER, S: "Real-time PCR detection of hemotropic mycoplasmas in healthy and unhealthy cats and dogs from the Barcelona area of Spain", JOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION, vol. 22, 2010, pages 270 - 274
SAMBROOK ET AL.: "Molecular Cloning, a laboratory manual", COLD SPRING HARBOR LABORAORY PRESS
STECK, T. L.: "The organization of proteins in the human red blood cell membrane. A review", J CELL BIOL, vol. 62, 1974, pages 1 - 19
SYKES, J. E., BAILIFF, N. L., BALL, L. M., FOREMAN, O., GEORGE, J. W., FRY, M. M.: "Identification of a novel hemotropic mycoplasma in a splenectomized dog with hemic neoplasia", JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION, vol. 224, 2004, pages 1946 - 1951
SYKES, J. E., DRAZENOVICH, N. L., BALL, L. M., LEUTENEGGER, C. M.: "Use of conventional and real-time polymerase chain reaction to determine the epidemiology of hemoplasma infections in anemic and nonanemic cats", JOURNAL OF VETERINARY INTERNAL MEDICINE, vol. 21, 2007, pages 685 - 693
TASKER, S., BINNS, S. H., DAY, M. J., GRUFFYDD-JONES, T. J., HARBOUR, D. A., HELPS, C. R., JENSEN, W. A., OLVER, C. S., LAPPIN, M.: "Use of a PCR assay to assess the prevalence and risk factors for Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum' in cats in the United Kingdom", VETERINARY RECORD, vol. 152, 2003, pages 193 - 198
TASKER, S., BRADDOCK, J. A., BARAL, R., HELPS, C. R., DAY, M. J., GRUFFYDD-JONES, T. J., MALIK, R.: "Diagnosis of feline haemoplasma infection in Australian cats using a real-time PCR assay", JOURNAL OF FELINE MEDICINE AND SURGERY, vol. 6, 2004, pages 345 - 354, XP004644817, DOI: doi:10.1016/j.jfms.2003.12.003
TASKER, S., CANEY, S. M., DAY, M. J., DEAN, R. S., HELPS, C. R., KNOWLES, T. G., LAIT, P. J., PINCHES, M. D., GRUFFYDD-JONES, T. J: "Effect of chronic feline immunodeficiency infection, and efficacy of marbofloxacin treatment, on 'Candidatus Mycoplasma haemominutum' infection", MICROBES AND INFECTION, vol. 8, 2006, pages 653 - 661, XP028072120, DOI: doi:10.1016/j.micinf.2005.08.015
TASKER, S., CANEY, S. M., DAY, M. J., DEAN, R. S., HELPS, C. R., KNOWLES, T. G., LAIT, P. J., PINCHES, M. D., GRUFFYDD-JONES, T. J: "Effect of chronic FIV infection, and efficacy of marbofloxacin treatment, on Mycoplasma haemofelis infection", VETERINARY MICROBIOLOGY, vol. 117, 2006, pages 169 - 179, XP024935675, DOI: doi:10.1016/j.vetmic.2006.06.015
TASKER, S., PETERS, . R., DAY, M. J., WILLI, B., HOFMANN-LEHMANN, R., GRUFFYDD-JONES, T. J., HELPS, C. R.: "Distribution of feline haemoplasmas in blood and tissue following experimental infection", MICROBIAL PATHOGENESIS, vol. 47, 2009, pages 334 - 340
TASKER, S., PETERS, . R., PAPASOULOTIS, K., CUE, S. M., WILLI, B., HOFMANN-LEHMANN, R., KNOWLES, T. G., GRUFFYDD-JONES, T. J., DAY: "Description of outcomes of experimental infection with feline haemoplasmas: haematology, Coombs' testing and blood glucose concentrations", VETERINARY MICROBIOLOGY, 2009
TASKER, S.: "Current concepts in feline haemobartonellosis", PRACTICE, vol. 28, 2006, pages 136 - 141
TASKER, S.: "Feline haemoplasmas - The past, present and future", 6TH EUROPEAN COLLEGE OF VETERINARY INTERNAL MEDICINE - COMPANION ANIMALS CONGRESS, 2006
TASKER, S.: "Haemotropic mycoplasmas: what's their real significance in cats?", JOURNAL OF FELINE MEDICINE AND SURGERY, vol. 12, 2010, pages 369 - 381
TURNER, C. M., BLOXHAM, P. A., COX, F. E., TURNER, C. B.: "Unreliable diagnosis of Haemobartonella felis", VETERINARY RECORD, vol. 119, 1986, pages 534 - 535
WENGI, N., WILLI, B., BORETTI, F. S., CATTORI, V., RIOND, B., MELI, M. L., REUSCH, C. E., LUTZ, H., HOFMANN-LEHMANN, R.: "Real-time PCR-based prevalence study, infection follow-up and molecular characterization of canine hemotropic mycoplasmas", VETERINARY MICROBIOLOGY, 2007
WENGI, N., WILLI, B., BORETTI, F. S., CATTORI, V., RIOND, B., MELI, M. L., REUSCH, C. E., LUTZ, H., HOFMANN-LEHMANN, R.: "Real-time PCR-based prevalence study, infection follow-up and molecular characterization of canine hemotropic mycoplasmas", VETERINARY MICROBIOLOGY, vol. 126, 2008, pages 132 - 141, XP022351070, DOI: doi:10.1016/j.vetmic.2007.06.018
WILLI, B., BORETTI, F. S., BAUMGARTNER, C., TASKER, S., WENGER, B., CATTORI, V., MELI, M. L., REUSCH, C. E., LUTZ, H., HOFMANN-LEH: "Prevalence, risk factor analysis, and follow-up of infections caused by three feline hemoplasma species in cats in Switzerland", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 44, 2006, pages 961 - 969, XP002425623, DOI: doi:10.1128/JCM.44.3.961-969.2006
WILLI, B., BORETTI, F. S., TASKER, S., MELI, M. L., WENGI, N., REUSCH, C. E., LUTZ, H., HOFMANN-LEHMANN, R.: "From Haemobartonella to hemoplasma: Molecular methods provide new insights", VETERINARY MICROBIOLOGY, vol. 125, 2007, pages 197 - 209, XP022306763, DOI: doi:10.1016/j.vetmic.2007.06.027
WILLI, B., TASKER, S., BORETTI, F. S., DOHERR, M. G., CATTORI, V., MELI, M. L., LOBETTI, R. G., MALIK, R., REUSCH, C. E., LUTZ, H.: "Phylogenetic analysis of 'Candidatus Mycoplasma turicensis' isolates from pet cats in the United Kingdom, Australia, and South Africa, with analysis of risk factors for infection", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 44, 2006, pages 4430 - 4435, XP009080625, DOI: doi:10.1128/JCM.00987-06

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