Assessment of Toll-like receptor 2, 4 and 9 SNP genotypes in canine sino-nasal aspergillosis.

BACKGROUND: The exact aetiology of canine sino-nasal aspergillosis (SNA) is unknown. In man, dysfunction in innate immunity, particularly in the function of pattern recognition receptors, is implicated in the pathogenesis of inflammatory sino-nasal disease and in fungal diseases. Associations between single nucleotide polymorphisms (SNPs) in Toll-like receptors (TLRs) and these diseases have been identified. Similarly, in dogs SNPs in genes encoding TLRs may be important in the pathogenesis of SNA. The aims of the present study were (1) to identify the presence of non-synonymous SNPs in the coding regions of the TLR2, 4 and 9 genes in dogs suffering from SNA, and (2) to investigate the SNP genotypes in dogs with SNA compared with a control population. RESULTS: Direct sequencing of nine dogs of various breeds with SNA revealed two non-synonymous SNPs in the coding region of TLR2, eight in TLR4 and four in TLR9. These non-synonymous SNPs were further evaluated in a case-control study of affected Golden Retrievers, Labrador Retrievers, Rottweilers and Beaucerons. Genotyping was performed using a combination of allele-specific primers and hydrolysis probe assays in 31 dogs with SNA and 31 controls. No significant difference in minor allele frequency was identified between these groups, for all studied SNPs, in any of the four breeds. CONCLUSIONS: These findings do not support a role for non-synonymous SNPs in the TLR 2, 4 and 9 coding regions in the pathogenesis of canine SNA, but do not exclude a role for innate immunity in the pathogenesis of the disease.

Genome sequence for "Candidatus Mycoplasma haemominutum," a low-pathogenicity hemoplasma species.

We present the genome sequence of "Candidatus Mycoplasma haemominutum" strain Birmingham 1, a low-pathogenicity feline hemoplasma strain.

Complete genome sequence of Mycoplasma haemofelis, a hemotropic mycoplasma.

Here, we present the genome sequence of Mycoplasma haemofelis strain Langford 1, representing the first hemotropic mycoplasma (hemoplasma) species to be completely sequenced and annotated. Originally isolated from a cat with hemolytic anemia, this strain induces severe hemolytic anemia when inoculated into specific-pathogen-free-derived cats. The genome sequence has provided insights into the biology of this uncultivatable hemoplasma and has identified potential molecular mechanisms underlying its pathogenicity.

Detection of feline haemoplasma species in experimental infections by in-situ hybridisation.

The aim of this study was to use fluorescence in-situ hybridisation (FISH) to search for the tissues and cell types important in survival and persistence of Mycoplasma haemofelis, "Candidatus Mycoplasma haemominutum" or "Candidatus Mycoplasma turicensis" in infected cats. A 16S rDNA probe for each species was applied to formalin-fixed, paraffin wax-embedded tissues sections collected from experimentally infected cats. Tissues (n = 12) were collected, at necropsy, from ten cats which had been infected with M. haemofelis, and one each with "Ca. M. haemominutum" and "Ca. M. turicensis". M. haemofelis specific hybridisation was present on red blood cells (RBCs) in all tissues from acutely infected cats, but not the majority of tissues from chronically infected cats. "Ca. M. haemominutum" specific hybridisation was present on scattered RBCs within the spleen and liver. Specific probe hybridisation was not detected in any of the "Ca. M. turicensis" infected tissues. Haemoplasmas were detected on the surface of RBCs only and not any other cell type. Additionally, FISH was limited by sensitivity and could not detect the lower numbers of organisms present in tissues of cats chronically infected with M. haemofelis. Occasional organisms were detected in cats acutely infected with "Ca. M. haemominutum" but not "Ca. M. turicensis".

Molecular characterization of the uncultivatable hemotropic bacterium Mycoplasma haemofelis.

Mycoplasma haemofelis is a pathogenic feline hemoplasma. Despite its importance, little is known about its metabolic pathways or mechanism of pathogenicity due to it being uncultivatable. The recently sequenced M. haemofelis str. Langford 1 genome was analysed and compared to those of other available hemoplasma genomes.Analysis showed that in hemoplasmas genes involved in carbohydrate metabolism are limited to enzymes of the glycolytic pathway, with glucose appearing to be the sole energy source. The majority of the pentose phosphate pathway enzymes that catalyze the de novo synthesis of ribonucleotides were absent, as were cell division protein FtsZ and chaperonins GroEL/ES. Uncharacterized protein paralogs containing putative surface expression motifs, comprised 62% of M. haemofelis and 19% of Mycoplasma suis genome coverage respectively, the majority of which were present in a small number of unstructured islands. Limited mass spectrometry and immunoblot data matched a number of characterized proteins and uncharacterized paralogs, confirming their expression and immunogenicity in vivo.These data have allowed further characterization of these important pathogens, including their limited metabolic capabilities, which may contribute to their uncultivatable status. A number of immunogenic proteins, and a potential mechanism for host immune system evasion, have been identified.

Genomic association and further characterisation of faecal immunoglobulin A deficiency in German Shepherd dogs.

BACKGROUND: Immunoglobulin A (IgA) deficiency, chronic enteropathies and exocrine pancreatic insufficiency (EPI) have a high prevalence in German Shepherd dogs (GSD). This prospective study determined the prevalence of faecal IgA deficiency (IgAD) in GSD and investigated several candidate genes and the canine genome for a region or locus co-segregating with IgAD in GSD. Faecal IgA concentrations were quantified and genomic DNA was extracted from 8 GSD with an undetectable faecal IgA (classified as IgAD) and 80 non-IgAD GSD. The canine minimal screening set II microsatellite markers were genotyped, with evidence of an association at p < 1.0 × 10-3 . Faecal IgA concentrations were also tested for an association with patient clinical and biochemical variables. RESULTS: Allele frequencies observed using the candidate gene approach were not associated with faecal IgAD in GSD. In the genome-wide association study (GWAS), the microsatellite marker FH2361 on canine chromosome 33 approached statistical significance for a link with IgAD in GSD (p = 1.2 × 10-3 ). A subsequent GWAS in 11 GSD with EPI and 80 control GSD revealed a significant association between EPI and FH2361 (p = 8.2 × 10-4 ). CONCLUSIONS: The lack of an association with the phenotype of faecal IgAD in GSD using the candidate gene approach and GWAS might suggests that faecal IgAD in GSD is a relative or transient state of deficiency. However, the prevalence of faecal IgAD in GSD appears to be low (<3%). The relationship between faecal IgAD, EPI and loci close to FH2361 on canine chromosome 33 in GSD warrants further investigation.

Prevalence of hemotropic mycoplasmas in healthy and unhealthy cats and dogs in Spain.

The aims of the present study were to determine the prevalence of hemoplasmas in cats and dogs from the Barcelona area of Spain with the use of species-specific quantitative polymerase chain reaction (qPCR) assays and to evaluate any associations between hemoplasma infection, clinical presentation, and vector-borne infections. Blood samples from cats (191) and dogs (182) were included and were classified as healthy (149) or unhealthy (224). Ethylenediamine tetra-acetic acid blood samples underwent DNA extraction and qPCR analysis. Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum', and 'Candidatus Mycoplasma turicensis' were detected in cats, whereas Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum' were detected in dogs, with prevalences of 3.7%, 9.9%, 0.5%, 14.3%, and 0.6%, respectively. In cats, no association between hemoplasma infection and health status, age, breed, presence of anemia, Feline leukemia virus status, and other vector-borne infections was found, but outdoor access (P = 0.009), male sex (P = 0.01), and Feline immunodeficiency virus status (P < 0.001) were significantly associated with hemoplasma infection. In dogs, sex, age, health status, presence of anemia, and breed were not significantly associated with hemoplasma infection, but a significant association was found between hemoplasma infection and vector-borne infections (P < 0.001). The present report documents the occurrence of feline 'Candidatus M. turicensis' and canine 'Candidatus M. haematoparvum' infections in Spain.

Ehrlichia canis infection in the cerebrospinal fluid of a dog characterized by morulae within monocytes and neutrophils.

An 8-year-old neutered female English Pointer was referred to a veterinary referral center (southwest of England) with a 4-5-month history of fecal incontinence and no evidence of urinary incontinence. Blood and free-catch urine samples were collected and sent to an off-site laboratory. Further investigations were postponed until laboratory results were available. Blood results showed a mild leukopenia, mild nonregenerative anemia, moderate to marked thrombocytopenia, and a mild increase in ALT and ALP activities. The primary veterinarian and client did not proceed with any further investigations for thrombocytopenia. Three weeks after the initial presentation, there was considerable clinical deterioration and progression of neurologic signs. Thoracic radiographs and an abdominal ultrasonographic examination were unremarkable. Magnetic resonance imaging (MRI) of the brain and spinal cord revealed an intramedullary lesion at the level of the C7 vertebra, a cystic lesion in the forebrain, and a bilateral lesion in the thalamus. A lumbar cerebrospinal fluid (CSF) was collected. CSF analysis showed a robustly increased protein concentration and marked pleocytosis. The cytologic evaluation revealed a mixed cellular population. Occasional neutrophils and monocytoid cells showed purple spherical intracellular inclusions, resembling Ehrlichia morulae. An aliquot of CSF was used off-label with a dot ELISA test, which showed a strong positive result for antibodies against Ehrlichia canis/Ehrlichia ewingii. PCR identified these morulae to be E canis. To best of the authors' knowledge, this is the first case of ehrlichial infection in canine CSF where Ehrlichia sub-species morulae present within neutrophils were confirmed to be Ehrlichia canis using PCR.

Distribution of Mycoplasma haemofelis in blood and tissues following experimental infection.

The aim of the study was to describe blood and tissue copy number distribution during Mycoplasma haemofelis infection and determine if sequestration of organisms in body tissues could explain blood copy number cycling in infected cats. Thirteen domestic-shorthaired cats were used. Blood samples were regularly collected, and at a differing time point post-infection for each cat, tissue samples also collected, for quantitative PCR (qPCR). Absolute haemoplasma copy numbers were calculated for all blood and tissue samples, as well as an estimation of the ratio of tissue haemoplasma copy number to that expected in the tissue if a positive qPCR result arose due to tissue blood supply alone. Cats with high or moderate M. haemofelis blood copy numbers at the time of tissue collection had fewer M. haemofelis copies in most tissues than expected due to the tissue blood supply alone; only splenic and lung tissues consistently contained more M. haemofelis. However tissues collected from cats at a time of very low M. haemofelis blood copy numbers, when putative copy number cycling nadirs were occurring, were usually qPCR negative. Hence no evidence of significant tissue M. haemofelis sequestration was found in this study to explain the copy number cycling reported with this feline haemoplasma species.

RNase P RNA gene (rnpB) phylogeny of Hemoplasmas and other Mycoplasma species.

Partial sequences of the RNase P RNA gene (rnpB) were obtained from a number of hemoplasmas and other Mycoplasma species. Phylogenetic analysis of these sequences showed that all hemoplasmas were present within a single clade and were most closely related to Mycoplasma fastidiosum, similar to the results found with 16S rRNA gene phylogeny.

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.

Three distinct species of feline haemoplasmas are recognised: Mycoplasma haemofelis (Mhf), Candidatus Mycoplasma haemominutum (CMhm) and Candidatus Mycoplasma turicensis (CMt). These species differ in pathogenicity as Mhf and CMt can be associated with anaemia whereas CMhm usually results in few clinical signs. The purpose of this study was to develop quantitative real-time PCR assays for the detection of all three feline haemoplasma species combined with an endogenous internal control and to determine the prevalence of infection, using these assays, in 1592 EDTA blood samples submitted to Langford Veterinary Diagnostics, University of Bristol for haemoplasma testing. Primers and TaqMan probes were designed against published 16S rDNA sequences. These assays were combined with a feline 28S rDNA-specific assay to produce three duplex assays. The assays detected 1-10 copies of a sequence-specific plasmid per PCR. None of the assays showed cross-reactivity with 10(6) copies of a sequence-specific plasmid from the non-target haemoplasma species. Real-time PCR was performed on all samples using the three assays. Seven samples were negative for feline 28S rDNA and were excluded from the study. Of the remaining 1585 samples, 45 (2.8%), 177 (11.2%) and 27 (1.7%) samples were positive for Mhf, CMhm and CMt, respectively, including 11 Mhf/CMhm, 10 CMhm/CMt and two Mhf/CMt dual infections and two triple infections. The results of this study demonstrate the utility of these new duplex PCR assays for the detection of haemoplasma infections. CMhm was the most common infection and CMt infections were often associated with co-infection with other haemoplasma species, especially CMhm.

Whole blood and tissue fungal DNA quantification in the diagnosis of canine sino-nasal aspergillosis.

Various combinations of tests are used to confirm the diagnosis of canine sino-nasal aspergillosis (SNA) because false-positive and false-negative results can occur with each test. Therefore, the aim of this study was to evaluate whether detection of fungal DNA in blood and nasal tissue samples was of value in the clinical diagnosis of this disease. Four groups were included in the study (dogs with SNA, lymphoplasmacytic rhinitis or nasal neoplasia, and control animals). Real-time PCR assays detecting DNA from all Penicillium and Aspergillus species (PenAsp assay) or species-specific DNA from A. fumigatus, A. terreus, A. flavus and A. niger were applied to whole blood and nasal tissue samples. Results obtained by PCR were compared between the groups. Sensitivity, specificity, positive and negative predictive values (PPV and NPV) for fungal DNA detection were compared with those for alternative diagnostic procedures including histopathology, serology and fungal culture. Significantly more fungal DNA was detected by the PenAsp assay in tissue biopsies from dogs with SNA than in the three other groups. Sensitivity, specificity, PPV and NPV for this method were 1.00, 0.06, 0.32 and 1.00. A. fumigatus DNA was detected in seven tissue biopsies from dogs with SNA and in one biopsy from a dog with a nasal tumour. Sensitivity, specificity, PPV and NPV for this diagnostic test were 0.50, 0.97, 0.87 and 0.82. No significant difference was found between the groups with respect to the amount of DNA detected in blood by the PenAsp assay. Sensitivity, specificity, PPV and NPV for this method were 0.71, 0.24, 0.31 and 0.64. A. fumigatus DNA was detected in the blood of three dogs with SNA and sixteen dogs without SNA. Sensitivity, specificity, PPV and NPV for this diagnostic tool were 0.21, 0.45, 0.15 and 0.54. Detection of A. fumigatus DNA in nasal tissue had the highest specificity, PPV and NPV but sensitivity of this method was low. Detection of fungal DNA in whole blood was of no value in the diagnosis of SNA.

Development and application of multiple internal reference (housekeeper) gene assays for accurate normalisation of canine gene expression studies.

Measurement of mRNA expression by real-time RT-PCR (QRT-PCR) has proven to be an important and powerful tool for the investigation of the pathogenesis of inflammatory and immune-mediated diseases in many species. This methodology has proven particularly valuable in the dog, a species for which there are currently few specific antibodies for measurement of relevant proteins. Internal control (housekeeper) mRNAs are widely used for normalisation of QRT-PCR results. The validation and use of multiple internal control mRNAs for increased accuracy of normalisation has been described for humans and rodents. The aims of this study were to develop QRT-PCR assays for 11 potential internal control mRNAs in the dog (ACTB, B(2)M, G3PDH, HMBS, HPRT1, RPL13A, RPL32, RPS18, SDHA, TBP and YWAZ) and validate their use with bone marrow, colon, duodenum, heart, kidney, liver, lung, lymph node, skeletal muscle, pancreas, spleen and stomach from seven dogs. Endoscopic biopsies of the superficial duodenal mucosa were also obtained from nine dogs suffering from chronic gastro-oesophageal disease. The most stably expressed genes varied in the tissues examined. RPL13A and RPL32 (both components of the 60S ribosomal subunit) were the most stably expressed genes in the majority of the tissues examined, whereas ACTB and B(2)M were the least stable. Distinct internal control genes were shown to be most appropriate for use in full-thickness versus superficial mucosal biopsies of the duodenum. The results of this study indicate that there are no universal control genes for gene expression studies in canine tissues. It is important to use multiple internal control genes based upon a survey of potential control genes applied to representative samples from different disease groups, culture conditions and/or time points in an experimental study.

Quantification of mRNA encoding cytokines and chemokines in nasal biopsies from dogs with sino-nasal aspergillosis.

Canine sino-nasal aspergillosis is usually caused by Aspergillus fumigatus and is similar to human chronic erosive non-invasive fungal sinusitis. The pathogenesis of the disease is poorly understood. We investigated the nature of the local immune response mounted in canine sino-nasal aspergillosis. Quantitative RT-PCR was carried out on RNA isolated from nasal biopsies from diseased and control dogs, using specific assays designed to amplify mRNA encoding a panel of cytokines and chemokines. Canine sino-nasal aspergillosis was associated with significantly increased expression of mRNA encoding MCP-1, -2, -3 and -4, IL-8, IL-10, IL-18 and TNF-alpha relative to controls (P<0.01) but there was no difference between groups with respect to IL-4, IL-5, IL-6, IL-12, TGF-beta, and eotaxin-2 and -3. The up-regulation of proinflammatory cytokines and chemokines related to the influx of phagocytic cells might account for the localisation of this infection to the upper respiratory tract. The up-regulation of the expression of the immunomodulatory cytokine IL-10 in nasal tissue from affected dogs might be important in limiting the extent of local tissue destruction, but might also account for the fact that infected dogs are generally unable to clear this infection spontaneously.

Immunopathogenic pathways in canine inflammatory myopathies resemble human myositis.

Progress in the treatment of inflammatory myopathies is impeded by the lack of suitable animal models. Inflammatory myopathies occur spontaneously in the dog, are a heterogeneous group of disorders, and are more common than in humans. Clinical signs of weakness and muscle atrophy are reliably present, and there are histological and immunohistological similarities to forms of human myositis. In this study, microarray technology followed by quantitative real-time PCR and immunohistochemistry on muscle biopsy sections was used to investigate gene expression in cases of canine inflammatory myopathies. Several genes involved with innate and adaptive immunity were highly upregulated including those that participate in macrophage and dendritic cell activation and migration, and antigen processing and presentation. Other genes including those that participate in B cell growth, development, migration and activation, immunoglobulin genes, genes in pro-inflammatory and anti-inflammatory pathways, and genes involved with tissue remodeling were upregulated. In previous reports utilizing microarray technology in human myositis, there was activation of similar pathways involved in the immune response. This study strengthens the argument that forms of canine myositis may be important animal models of human myositis and suggests useful biomarkers for therapeutic response using the dog in pre-clinical trials.

Cytokine mRNA quantification in duodenal mucosa from dogs with chronic enteropathies by real-time reverse transcriptase polymerase chain reaction.

The pathogenesis of inflammatory bowel disease (IBD) and antibiotic-responsive diarrhea (ARD) in dogs likely involves an interaction between the intestinal immune system and luminal bacterial or food antigens. German Shepherd Dogs (GSD) are particularly predisposed to both IBD and ARD. CD4+ T cells are important for the regulation of immune responses in the mucosa, and they exert their effects through the secretion of cytokines. The present study examined the role of cytokines in the pathogenesis of canine chronic enteropathies by quantification of mRNA encoding interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, interferon gamma, tumor necrosis factor-alpha, transforming growth factor-beta, and glyceraldehyde-3-phosphate dehydrogenase by real-time reverse transcriptase polymerase chain reaction in duodenal mucosal biopsies obtained from 39 dogs with chronic diarrhea and 18 control dogs. Contemporaneously collected biopsies were assessed for histologic changes with a 4-point grading system. No significant difference in the expression of cytokine mRNA (P > .01) was detected between dogs with and those without chronic diarrhea. Similarly, no significant differences in cytokine mRNA expression were observed between GSD and other breeds with chronic diarrhea, or between histologically normal duodenal mucosa and that with evidence of inflammatory change. Failure to detect a difference in mRNA expression does not rule out the possibility of a defect downstream at the level of translation or protein function. No conclusion can be drawn from these data as to the predominant CD4+ cell type in the pathogenesis of these canine chronic enteropathies.

Measurement of messenger RNA encoding the alpha-chain, polymeric immunoglobulin receptor, and J-chain in duodenal mucosa from dogs with and without chronic diarrhea by use of quantitative real-time reverse transcription-polymerase chain reaction assays.

OBJECTIVE: To examine the difference in expression of messenger RNA (mRNA) transcripts for polymeric immunoglobulin receptor (plgR), alpha-chain, and J-chain determined by use of quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) assays in duodenal biopsy specimens obtained from dogs with and without chronic diarrhea. SAMPLE POPULATION: Biopsy specimens of the proximal portion of the duodenum were obtained endoscopically from 39 dogs evaluated because of chronic diarrhea (12 German Shepherd Dogs and 27 non-German Shepherd Dog breeds); specimens were also obtained from a control group of 7 dogs evaluated because of other gastrointestinal tract diseases and 2 dogs that were euthanatized as a result of nongastrointestinal tract disease. PROCEDURE: Dogs were anesthetized, and multiple mucosal biopsy specimens were obtained endoscopically at the level of the caudal duodenal flexure by use of biopsy forceps; in 2 control dogs, samples were obtained from the descending duodenum within 5 minutes of euthanasia. One-step QRT-PCR was used to quantify the level of expression of transcripts for the housekeeper gene glyceraldehyde-3-phosphate dehydrogenase, plgR, alpha-chain, and J-chain in duodenal mucosal tissue. RESULTS: There was no significant difference in the level of expression of any transcript among non-German Shepherd Dog breeds without diarrhea (control group), non-German Shepherd Dog breeds with chronic diarrhea, and German Shepherd Dogs with chronic diarrhea. Conclusions and Clinical Relevance-Results indicated that the susceptibility of German Shepherd Dogs to chronic diarrhea is not a result of simple failure of transcription of the key genes that encode molecules involved in mucosal IgA secretion.

Quantitative real-time RT-PCR measurement of mRNA encoding alpha-chain, pIgR and J-chain from canine duodenal mucosa.

IgA is the predominant immunoglobulin class in mucosal secretions and secretory deficiencies may predispose to chronic enteropathies. The polymeric immunoglobulin receptor (pIgR) facilitates the transport of IgA across the epithelial border. Critical to the transport of IgA by pIgR is the presence of a polypeptide joining chain (J-chain) linking the IgA monomers of the dimeric IgA molecule. In this study we examine the difference in expression of mRNA transcripts for pIgR, alpha-chain and J-chain by real-time reverse-transcription polymerase chain reaction (RT-PCR) in endoscopic biopsies from the duodenum of dogs with and without chronic diarrhoea. One-step, real-time RT-PCR was used to quantify the level of expression of transcripts for the housekeeper gene G3PDH, pIgR, alpha-chain and J-chain. There was no significant difference in expression of any transcript between dogs with (n=11) and without (n=8) chronic diarrhoea. Expression of alpha-chain mRNA in both groups had a similar bimodal distribution, as individuals either expressed relatively 'high' or 'low' levels of this transcript. The secretion of IgA by plasma cells is under the control of Th-2 cytokines, therefore the finding of 'high' and 'low' levels of alpha-chain expression may reflect different levels of these cytokines in duodenal mucosa.

A novel haemoplasma species identified in archived primate blood smears.

In order to confirm a microscopic diagnosis of 'eperythrozoonosis' made over 40 years ago in a captive owl monkey (Aotus trivirgatus), DNA was extracted from archived fixed and stained blood smears and subjected to generic haemotropic mycoplasma (haemoplasma) quantitative real-time PCR (qPCR) and a human glyceraldehyde-3-phosphate dehydrogenase qPCR as an amplification control. The qPCRs confirmed the extraction of host DNA from the samples and the presence of a haemoplasma species. Partial 16S rRNA and ribonuclease P ribosomal gene fragments were amplified by PCR, cloned and sequenced. Sequence data and phylogeny showed the owl monkey haemoplasma to lie in the haemominutum clade of haemoplasmas, most closely related to 'Candidatus Mycoplasma kahaneii'. This study confirms the use of generic haemoplasma qPCRs to successfully amplify haemoplasma DNA from fixed, stained and archived blood smears from the early 1970s and provides molecular confirmation of the existence of a novel haemoplasma species in an owl monkey, for which the name 'Candidatus Mycoplasma aoti' sp. nov. is proposed.

Antigen specificity of the humoral immune response to Mycoplasma haemofelis infection.

The aim of the present study was to characterize the antigenic specificity of the humoral immune response made by cats infected with the feline hemoplasma, Mycoplasma haemofelis. A crude M. haemofelis antigen preparation was prepared from red blood cells (RBCs) collected from a cat at the time of a high level of bacteremia. Plasma samples were collected from six cats before and after experimental infection with M. haemofelis, with regular sampling being performed from 15 to 149 or 153 days postinfection (dpi). Preinfection RBC membrane ghosts were prepared from these six cats and used to identify erythrocyte proteins that may have contaminated the M. haemofelis antigen preparation. The M. haemofelis antigen preparation comprised 11 protein bands. The immunodominant bands on Western blotting with infected cat plasma had molecular masses of 78, 68, 60, 48, and 38 kDa. Most cats (n = 5) had plasma antibody that reacted with at least one band (always including the one of 68 kDa) at 15 dpi, and all cats were seroreactive by 29 dpi. The maximum number of antibodies from an individual animal specific for an antigen was identified in plasma collected from 57 to 99 dpi. Contamination of the M. haemofelis antigen preparation with RBC membrane proteins was observed. The contaminating RBC proteins had molecular masses of from 71 to 72 kDa (consistent with band 4.2) and 261 and 238 kDa (consistent with spectrin), and these were recognized by all plasma samples. A range of M. haemofelis antigens is recognized 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.