Evaluating the association between blood genotype or phenotype and haemoplasma infection in UK and Italian cats.

BACKGROUND: In humans, blood groups are associated with varying prevalence of infections. The aim of this study was to determine if associations exist between the feline AB blood group system and haemoplasma infection. METHODS: Data from two studies were combined. In the first study, DNA samples from 131 haemoplasma-infected and 132 haemoplasma-uninfected UK cats underwent pyrosequencing to determine their blood genotype as AA, Ab or bb. In the second study, blood samples from 160 Italian cats of known blood phenotype A, B or AB underwent PCR testing for feline haemoplasma species DNA. RESULTS: Haemoplasma infection was demonstrated in cats of all phenotypes and genotypes. A significantly higher number of Ab genotype cats tested positive for overall haemoplasma infection status (p = 0.04) and for Mycoplasma haemofelis infection (p = 0.03). LIMITATIONS: Haemoplasma-infected Italian cats were few, possibly increasing the chance of type II error, and the presence of purebred cats in the sample population may have had a confounding effect. CONCLUSIONS: Feline haemoplasmas do not appear to preferentially use either blood type A or B antigens as attachment sites for erythrocyte colonisation. Further investigations in a larger number of haemoplasma-infected cats of known blood phenotype are warranted to explain the association between genotype Ab and haemoplasma infection.

Prevalence and risk factor analysis for feline haemoplasmas in cats from Northern Serbia, with molecular subtyping of feline immunodeficiency virus.

OBJECTIVES: The objectives of this study were to estimate the prevalence of feline haemoplasma infections in Northern Serbia, identify potential risk factors and perform molecular subtyping of feline immunodeficiency virus (FIV). METHODS: PCR analysis for feline haemoplasmas was performed on surplus EDTA blood samples from 373 cats from the Belgrade region, Serbia. An ELISA was used to determine the prevalence of feline leukaemia virus (FeLV) and FIV; PCR was performed on a subpopulation of these cats. FIV subtyping was performed using PCR. RESULTS: Within this population, 64/373 cats (17.2%) were infected with one or more haemoplasma species. Mycoplasma haemofelis was detected in 20/373 cats (5.4%), 'Candidatus Mycoplasma haemominutum' in 47/373 cats (12.6%) and 'Candidatus Mycoplasma turicensis' in 23/373 cats (6.2%). Coinfections were observed in 21/373 cats (5.6%). Based on ELISA serological retroviral testing, 4/310 cats (1.3%) were infected with FeLV, whereas 78/331 (23.6%) were infected with FIV. Multivariable analysis identified significant associations between haemoplasma infection and anaemia (anaemic/non-anaemic, odds ratio [OR] 2.7, 95% confidence interval [CI] 1.04-7.1; P = 0.041]), male gender (male/female, OR 4.5, 95% CI 2.22-9.03; P <0.0005), outdoor access (yes/no, OR 5.2, 95% CI 2.28-11.92; P <0.0005), non-pedigree breed (non-pedigree/pedigree, OR 5.5, 95% CI 1.24-24.84; P = 0.025) and FIV seropositive status (positive/negative, OR 2.4, 95% CI 1.21-4.83; P = 0.012). PCR analysis of the FIV ELISA-positive samples revealed clade D as being the most prevalent. CONCLUSIONS AND RELEVANCE: All three known species of feline haemoplasma were detected, confirming their presence in Serbia; 'Candidatus Mycoplasma haemominutum' was the most prevalent. We found a high prevalence of FIV-infected cats and FIV clade D was most prevalent.

Anaplasma phagocytophilum, Bartonella spp., haemoplasma species and Hepatozoon spp. in ticks infesting cats: a large-scale survey.

BACKGROUND: Ticks derived from cats have rarely been evaluated for the presence of pathogens. The aim of this study was to determine the prevalence of Anaplasma phagocytophilum, Bartonella spp., haemoplasma species and Hepatozoon spp. in ticks collected from cats in the UK. METHODS: Five hundred and forty DNA samples extracted from 540 ticks collected from cats presenting to veterinarians in UK practices were used. Samples underwent a conventional generic PCR assay for detection of Hepatozoon spp. and real-time quantitative PCR assays for detection of Anaplasma phagocytophilum and three feline haemoplasma species and a generic qPCR for detection of Bartonella spp. Feline 28S rDNA served as an endogenous internal PCR control and was assessed within the haemoplasma qPCR assays. Samples positive on the conventional and quantitative generic PCRs were submitted for DNA sequencing for species identification. RESULTS: Feline 28S rDNA was amplified from 475 of the 540 (88.0%) ticks. No evidence of PCR inhibition was found using an internal amplification control. Of 540 ticks, 19 (3.5%) contained DNA from one of the tick-borne pathogens evaluated. Pathogens detected were: A. phagocytophilum (n = 5; 0.9%), Bartonella spp. (n = 7; 1.3%) [including Bartonella henselae (n = 3; 0.6%) and Bartonella clarridgeiae (n = 1; 0.2%)], haemoplasma species (n = 5; 0.9%), "Candidatus Mycoplasma haemominutum" (n = 3; 0.6%), Mycoplasma haemofelis (n = 1; 0.2%), "Candidatus Mycoplasma turicensis" (n = 1; 0.2%), Hepatozoon spp. (n = 2; 0.4%), Hepatozoon felis (n = 1; 0.2%) and Hepatozoon silvestris (n = 1; 0.2%). CONCLUSION: These data provide important information on the prevalence of tick-borne pathogens in ticks infesting cats, with the identification of haemoplasma species, A. phagocytophilum, H. felis and Bartonella spp. (including B. henselae and B. clarridgeiae). This study also documents the first report of H. silvestris in ticks collected from domestic cats.

Correction: A Novel Variant in CMAH Is Associated with Blood Type AB in Ragdoll Cats.

[This corrects the article DOI: 10.1371/journal.pone.0154973.].

Limitations of using feline coronavirus spike protein gene mutations to diagnose feline infectious peritonitis.

Feline infectious peritonitis (FIP) is a fatal disease of cats, and a sequela of systemic feline coronavirus (FCoV) infection. Mutations in the viral spike (S) gene have been associated with FCoVs found in tissues from cats with FIP, but not FCoVs found in faeces from healthy cats, and are implicated in monocyte/macrophage tropism and systemic spread. This study was designed to determine whether S gene mutation analysis can reliably diagnose FIP. Cats were categorised as with FIP (n = 57) or without FIP (n = 45) based on gross post-mortem and histopathological examination including immunohistochemistry for FCoV antigen. RNA was purified from available tissue, fluid and faeces. Reverse-transcriptase quantitative-PCR (RT-qPCR) was performed on all samples using FCoV-specific primers, followed by sequencing of a section of the S gene on RT-qPCR positive samples. Samples were available from a total of 102 cats. Tissue, fluid, and faecal samples from cats with FIP were more likely to be FCoV RT-qPCR-positive (90.4, 78.4 and 64.6% respectively) than those from cats without FIP (7.8, 2.1 and 20% respectively). Identification of S gene mutated FCoVs as an additional step to the detection of FCoV alone, only moderately increased specificity for tissue samples (from 92.6 to 94.6%) but specificity was unchanged for fluid samples (97.9%) for FIP diagnosis; however, sensitivity was markedly decreased for tissue (from 89.8 to 80.9%) and fluid samples (from 78.4 to 60%) for FIP diagnosis. These findings demonstrate that S gene mutation analysis in FCoVs does not substantially improve the ability to diagnose FIP as compared to detection of FCoV alone.

Frequency, clinicopathological features and phylogenetic analysis of feline morbillivirus in cats in Istanbul, Turkey.

Objectives The aim of the study was to investigate feline morbillivirus (FmoPV) frequency, phylogeny and associated pathology in cats in Istanbul, Turkey. Methods Samples from sick (n = 96) and dead ( n = 15) cats were analysed using reverse transcription PCR. Blood and urine analyses and histopathology were also performed. Results FmoPV RNA was detected in six cats (5.4%), including three sick (in the urine) and three dead cats (tissues). A significantly greater proportion of FmoPV RNA-positive cats had street access compared with non-infected cats. Blood samples from the morbillivirus-positive cats were negative for morbillivirus RNA. Tubular parenchymal cells, lymphoid and plasma cells in kidney and hepatocytes, lymphoid and plasma cells in liver from dead cats were also positive by immunohistochemistry for the viral N protein. Two FmoPV-positive cats were also positive for feline coronavirus RNA and one cat for feline immunodeficiency virus RNA and feline leukaemia virus proviral DNA. Phylogenetic analysis of the six FmoPV-positive cats showed that the strains were grouped into cluster D and had high similarity (98.5-100%) with strains from Japan and Germany. In the three FmoPV RNA-positive sick cats, respiratory, urinary and digestive system signs were observed as well as weight loss, fever and depression in some cats. Similar clinical signs were also seen in the morbillivirus RNA-negative sick cats. FmoPV RNA-positive cats had lower median red blood cell count, haemoglobin, albumin, albumin/globulin and urobilinogen and higher alanine transaminase, alkaline phosphatase and bilirubin compared with non-infected cats. Significant histopathology of FmoPV RNA-positive dead cats included tubulointerstitial nephritis characterised by severe granular and vacuolar degeneration of the epithelial cells of the cortical and medullary tubules as well as mononuclear cell infiltrates. Widespread lymphoid cell infiltrates were detected in the renal cortex and medullary regions of the kidneys. Cellular infiltration, cholangiohepatitis and focal necrosis in the liver were also found. Although virus-infected cells were found in the kidney and liver of FmoRV RNA-positive cats, tubulointerstitial nephritis, cholangiohepatitis and focal necrosis seen in FmoRV RNA-positive cats were similar to those observed in FmoRV RNA-negative cats. Conclusions and relevance This is the first study to show the presence of FmoPV infection in cats in Turkey. Sick cats, particularly those with kidney disease, should be tested for this virus. The genotypes found in this study were similar to previously reported strains, indicating that circulating morbilliviruses in Turkey are conserved.

A Novel Variant in CMAH Is Associated with Blood Type AB in Ragdoll Cats.

The enzyme cytidine monophospho-N-acetylneuraminic acid hydroxylase is associated with the production of sialic acids on cat red blood cells. The cat has one major blood group with three serotypes; the most common blood type A being dominant to type B. A third rare blood type is known as AB and has an unclear mode of inheritance. Cat blood type antigens are defined, with N-glycolylneuraminic acid being associated with type A and N-acetylneuraminic acid with type B. Blood type AB is serologically characterized by agglutination using typing reagents directed against both A and B epitopes. While a genetic characterization of blood type B has been achieved, the rare type AB serotype remains genetically uncharacterized. A genome-wide association study in Ragdoll cats (22 cases and 15 controls) detected a significant association between blood type AB and SNPs on cat chromosome B2, with the most highly associated SNP being at position 4,487,432 near the candidate gene cytidine monophospho-N-acetylneuraminic acid hydroxylase. A novel variant, c.364C>T, was identified that is highly associated with blood type AB in Ragdoll cats and, to a lesser degree, with type AB in random bred cats. The newly identified variant is probably linked with blood type AB in Ragdoll cats, and is associated with the expression of both antigens (N-glycolylneuraminic acid and N-acetylneuraminic acid) on the red blood cell membrane. Other variants, not identified by this work, are likely to be associated with blood type AB in other breeds of cat.

Prevalence and phylogenetic analysis of haemoplasmas from cats infected with multiple species.

Mycoplasma haemofelis (Mhf), 'Candidatus Mycoplasma haemominutum' (CMhm) and 'Candidatus Mycoplasma turicensis' (CMt) are agents of feline haemoplasmosis and can induce anaemia in cats. This study aimed to determine the prevalence and phylogeny of haemoplasma species in cats from Brazil's capital and surrounding areas, and whether correlation with haematological abnormalities existed. Feline haemoplasmas were found in 13.8% of 432 cats. CMhm was the most prevalent species (in 13.8% of cats), followed by Mhf (11.1%) and CMt (4.4%). Over 80% of haemoplasma-infected cats harboured two or more feline haemoplasma species: 7.1% of cats were co-infected with Mhf/CMhm, 0.4% with CMhm/CMt and 3.9% with Mhf/CMhm/CMt. Male gender was significantly associated with haemoplasma infections. No association was found between qPCR haemoplasma status and haematological variables, however CMhm relative copy numbers were correlated with red blood cell (RBC) numbers and packed cell volume (PCV). Haemoplasma 16S rRNA gene sequences (> 1 Kb) were derived from co-infected cats using novel haemoplasma species-specific primers. This allowed 16S rRNA gene sequences to be obtained despite the high level of co-infection, which precluded the use of universal 16S rRNA gene primers. Within each species, the Mhf, CMhm and CMt sequences showed > 99.8%, > 98.5% and > 98.8% identity, respectively. The Mhf, CMhm and CMt sequences showed > 99.2%, > 98.4% and > 97.8% identity, respectively, with GenBank sequences. Phylogenetic analysis showed all Mhf sequences to reside in a single clade, whereas the CMhm and CMt sequences each grouped into three distinct subclades. These phylogeny findings suggest the existence of different CMhm and CMt strains.

Erythrocyte pyruvate kinase deficiency mutation identified in multiple breeds of domestic cats.

BACKGROUND: Erythrocyte pyruvate kinase deficiency (PK deficiency) is an inherited hemolytic anemia that has been documented in the Abyssinian and Somali breeds as well as random bred domestic shorthair cats. The disease results from mutations in PKLR, the gene encoding the regulatory glycolytic enzyme pyruvate kinase (PK). Multiple isozymes are produced by tissue-specific differential processing of PKLR mRNA. Perturbation of PK decreases erythrocyte longevity resulting in anemia. Additional signs include: severe lethargy, weakness, weight loss, jaundice, and abdominal enlargement. In domestic cats, PK deficiency has an autosomal recessive mode of inheritance with high variability in onset and severity of clinical symptoms. RESULTS: Sequence analysis of PKLR revealed an intron 5 single nucleotide polymorphism (SNP) at position 304 concordant with the disease phenotype in Abyssinian and Somali cats. Located 53 nucleotides upstream of the exon 6 splice site, cats with this SNP produce liver and blood processed mRNA with a 13 bp deletion at the 3' end of exon 5. The frame-shift mutation creates a stop codon at amino acid position 248 in exon 6. The frequency of the intronic SNP in 14,179 American and European cats representing 38 breeds, 76 western random bred cats and 111 cats of unknown breed is 6.31% and 9.35% when restricted to the 15 groups carrying the concordant SNP. CONCLUSIONS: PK testing is recommended for Bengals, Egyptian Maus, La Perms, Maine Coon cats, Norwegian Forest cats, Savannahs, Siberians, and Singapuras, in addition to Abyssinians and Somalis as well an any new breeds using the afore mentioned breeds in out crossing or development programs.

Haemoparasites of free-roaming dogs associated with several remote Aboriginal communities in Australia.

BACKGROUND: Tick-borne haemoparasites Babesia vogeli and Anaplasma platys are common among the free-roaming canine populations associated with Aboriginal communities in Australia, whilst the prevalence of haemoplasmas, which are also suspected to be tick-borne, remained unexplored. The aim of this study was to determine the prevalence of haemoplasma infection in these populations, and to identify any correlation with other haemoparasites. Blood was collected from 39 dogs associated with four Aboriginal communities and screened for infection using PCR and serology. DNA was purified and PCR analyses for piroplasms, Anaplasmataceae family bacteria and haemoplasmas performed. Serum was analysed using a commercial haemoparasite ELISA. Prevalence of infection was compared between communities. RESULTS: Seventeen dogs (44%) were infected (PCR positive) with Mycoplasma haemocanis, eight (21%) with 'Candidatus Mycoplasma haematoparvum', 20 (51%) with A. platys, and 17 (44%) with B. vogeli. Two dogs were infected with a novel haemoplasma as determined by DNA amplification and sequencing. Two dogs (5%) were serologically positive for Dirofilaria immitis antigens, one (3%) was positive for Ehrlichia canis antibodies and nine (24nbsp;%) were positive for A. platys antibodies. Co-infections were frequent. Haemoplasma prevalence was highest (73%, 16/22) in Central Australia and lowest (22%, 2/9) in Western Australia (p = 0.017). In contrast, B. vogeli prevalence was low in Central Australia (18%, 4/22) but higher (78%, 7/9) in Western Australia (p = 0.003). CONCLUSIONS: This is the first time haemoplasma infections, including a novel species, have been molecularly documented in Australian dogs. The wide regional variation in prevalence of some of the haemoparasite infections detected in this study warrants further investigation.

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.

First WNK4-hypokalemia animal model identified by genome-wide association in Burmese cats.

Burmese is an old and popular cat breed, however, several health concerns, such as hypokalemia and a craniofacial defect, are prevalent, endangering the general health of the breed. Hypokalemia, a subnormal serum potassium ion concentration ([K(+)]), most often occurs as a secondary problem but can occur as a primary problem, such as hypokalaemic periodic paralysis in humans, and as feline hypokalaemic periodic polymyopathy primarily in Burmese. The most characteristic clinical sign of hypokalemia in Burmese is a skeletal muscle weakness that is frequently episodic in nature, either generalized, or sometimes localized to the cervical and thoracic limb girdle muscles. Burmese hypokalemia is suspected to be a single locus autosomal recessive trait. A genome wide case-control study using the illumina Infinium Feline 63K iSelect DNA array was performed using 35 cases and 25 controls from the Burmese breed that identified a locus on chromosome E1 associated with hypokalemia. Within approximately 1.2 Mb of the highest associated SNP, two candidate genes were identified, KCNH4 and WNK4. Direct sequencing of the genes revealed a nonsense mutation, producing a premature stop codon within WNK4 (c.2899C>T), leading to a truncated protein that lacks the C-terminal coiled-coil domain and the highly conserved Akt1/SGK phosphorylation site. All cases were homozygous for the mutation. Although the exact mechanism causing hypokalemia has not been determined, extrapolation from the homologous human and mouse genes suggests the mechanism may involve a potassium-losing nephropathy. A genetic test to screen for the genetic defect within the active breeding population has been developed, which should lead to eradication of the mutation and improved general health within the breed. Moreover, the identified mutation may help clarify the role of the protein in K⁺ regulation and the cat represents the first animal model for WNK4-associated hypokalemia.

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.

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.

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.

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".

Detection of humoral response using a recombinant heat shock protein 70, DnaK, of Mycoplasma haemofelis in experimentally and naturally hemoplasma-infected cats.

Hemoplasmas is the trivial name given to a group of erythrocyte-parasitizing bacteria of the genus Mycoplasma. Of the feline hemoplasmas, Mycoplasma haemofelis is the most pathogenic, while "Candidatus Mycoplasma haemominutum" and "Candidatus Mycoplasma turicensis" are less pathogenic. Shotgun libraries of fragmented M. haemofelis genomic DNA were constructed, and random colonies were selected for DNA sequencing. In silico-translated amino acid sequences of putative open reading frames were compared to mass spectrometry data from M. haemofelis protein spots identified as being immunogenic by two-dimensional gel electrophoresis and Western blotting. Three of the spots matched the predicted sequences of a heat shock protein 70 (DnaK) homolog, elongation factor Ts, and a fragment of phosphoglycerate kinase found during library screening. A full-length copy of the M. haemofelis dnaK gene was cloned into Escherichia coli and recombinantly expressed. Recombinant M. haemofelis DnaK was purified and then used in Western blotting and an enzyme-linked immunosorbent assay (ELISA) to investigate the humoral immune response during acute infection in cats experimentally infected with M. haemofelis, "Ca. Mycoplasma haemominutum," or "Ca. Mycoplasma turicensis". The recombinant M. haemofelis DnaK ELISA also was used to screen clinical samples submitted for hemoplasma PCR testing to a commercial laboratory (n = 254). Experimentally infected cats became seropositive following infection, with a greater and earlier antibody response seen in cats inoculated with M. haemofelis than those seen in cats inoculated with "Ca. Mycoplasma haemominutum" or "Ca. Mycoplasma turicensis," by both Western blotting and ELISA. Of the clinical samples, 31.1% had antibodies detected by the ELISA but only 9.8% were positive by PCR for one or more hemoplasmas.

Investigation of human haemotropic Mycoplasma infections using a novel generic haemoplasma qPCR assay on blood samples and blood smears.

The aim of this study was to develop quantitative real-time (q)PCR assays to detect all known haemoplasma species, and a human housekeeping gene in order to demonstrate both successful DNA extraction from clinical samples and to test for sample inhibition, and to apply these qPCRs to human blood samples and blood smears. Sensitive and specific generic haemoplasma qPCR assays were developed to amplify haemoplasma species, as well as human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal amplification control. An optimized technique for extracting DNA from stained blood smears was also developed. These methods were applied to anonymized blood samples obtained from 100 human immunodeficiency virus (HIV)-infected South Africans and 920 UK patients undergoing haematological examination, and to 15 blood smears recruited from previous studies describing human haemoplasmosis. Human GAPDH levels were acceptable in all but three of the blood samples and all but two of the blood smears. The latter could have arisen due to DNA degradation due to the old age (over 35 years) of these smears. Haemoplasma infection was found in one HIV-infected South African, but the species could not be characterized due to the very low levels of DNA present. This report describes novel extraction and qPCR methodologies for haemoplasma screening. Previously reported human haemoplasmosis based on cytological diagnosis alone should be viewed with caution.

Use of real-time quantitative polymerase chain reaction to monitor antibiotic therapy in a dog with naturally acquired Mycoplasma haemocanis infection.

Mycoplasma haemocanis is a hemotropic bacterium that can be associated with acute hemolytic disease in immunocompromised or splenectomized dogs. The present case report describes for the first time the use of real-time quantitative polymerase chain reaction (qPCR) to monitor M. haemocanis infection in a splenectomized dog. The report also describes the application of real-time qPCR for the analysis of deoxyribonucleic acid extracted from stained blood films. The analysis of blood films from the time of initial presentation allowed a retrospective confirmation of M. haemocanis infection. The M. haemocanis copy numbers remained high throughout antibiotic treatment of this dog. A decline in copy numbers was only recorded after 11 months of therapy, when improvements in clinical and hematological indices were also noted. Clearance of infection was not achieved, and the dog remained positive for M. haemocanis at 3.5 months postcessation of antibiotic therapy. Cytological examination of blood films for the presence of organisms was insensitive for the detection of parasitemia.

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.