Prevalence and risk factors for common respiratory pathogens within a cohort of pet cats in the UK.

OBJECTIVES: Feline herpesvirus (FHV), feline calicivirus (FCV) and Chlamydia felis are common causes of upper respiratory tract disease (URTD) in cats. Their prevalence in the UK pet cat population has not been reported and little is known regarding the risk factors for their oral carriage. METHODS: Total nucleic acid was extracted from owner-collected buccal swabs (n=600) from cats enrolled in a self-selected longitudinal cohort study. Duplex quantitative PCRs for the detection of FHV and C. felis genomic DNA and reverse-transcriptase quantitative PCRs for the detection of FCV genomic RNA were performed. Duplicates, swabs with insufficient host DNA/RNA, and cats with missing data were excluded. Selected epidemiological data were interrogated using univariable and multi-variable logistic regression modelling to identify risk factors. RESULTS: Data from 430 cats were included in the final statistical model. Of these, 2.1% (n=9/430; 95% CI 1.0% to 3.9%) were positive for FHV, 13.3% (n=57/430; 95% CI 10.2% to 16.8%) positive for FCV and 1.2% (n=5/430; 95% CI 0.4% to 2.7%) positive for C. felis. FCV co-infection was present in five (44%) FHV-positive cats and three (60%) C. felis-positive cats. FCV carriage was more frequent in purebred cats (odds ratio 2.48; 95% CI 1.37 to 4.49) and in cats with current or historical clinical signs compatible with URTD (odds ratio 2.98; 95% CI 1.22 to 7.27). CLINICAL SIGNIFICANCE: FCV was the most frequently encountered URTD pathogen in this sample of cats; this should be noted for disinfectant choice. In cats suspected of having FHV or C. felis infection, assessment for co-infection with FCV is recommended.

Thyroid and renal function in cats following low-dose radioiodine (111Mbq) therapy.

OBJECTIVES: To describe the effect of low-dose (111MBq) radioiodine therapy on thyroid and renal function in hyperthyroid cats over a 12-month follow-up period. MATERIALS AND METHODS: Client-owned hyperthyroid cats underwent low-dose radioiodine therapy and were followed-up for 12 months. Immediately before radioiodine treatment, and at 1, 6 and 12 months afterwards, total thyroxine, thyroid stimulating hormone, serum creatinine and glomerular filtration rate were measured. RESULTS: Fifteen of the 24 (63%) cats achieved euthyroidism following low-dose radioiodine treatment. The incidence of overt hypothyroidism was six of 24 (25%) cats. Of the six cats developing overt hypothyroidism, three had decreased renal function, with decreased glomerular filtration rate preceding azotaemia in two of these individuals. Transient overt or subclinical hypothyroidism before restoration of euthyroidism was not observed. CLINICAL SIGNIFICANCE: Low-dose radioiodine is effective treatment for hyperthyroidism in most cats but overt hypothyroidism may develop in some. Concurrent early decline in renal function may only be detected by measuring glomerular filtration rate rather than serum creatinine in some cats. Monitoring following radioiodine treatment should include total thyroxine and thyroid stimulating hormone and measurement of glomerular filtration rate should be considered in non-azotaemic cats.

Inflammatory Mediators in the Mesenteric Lymph Nodes, Site of a Possible Intermediate Phase in the Immune Response to Feline Coronavirus and the Pathogenesis of Feline Infectious Peritonitis?

Feline infectious peritonitis (FIP) is an almost invariably fatal feline coronavirus (FCoV)-induced disease thought to arise from a combination of viral mutations and an overexuberant immune response. Natural initial enteric FCoV infection may remain subclinical, or result in mild enteric signs or the development of FIP; cats may also carry the virus systemically with no adverse effect. This study screened mesenteric lymph nodes (MLNs), the presumed first site of FCoV spread from the intestine regardless of viraemia, for changes in the transcription of a panel of innate immune response mediators in response to systemic FCoV infection and with FIP, aiming to identify key pathways triggered by FCoV. Cats with and without FIP, the latter with and without FCoV infection in the MLN, were compared. Higher expression levels in FIP were found for toll-like receptors (TLRs) 2, 4 and 8. These are part of the first line of defence and suggest a response to both viral structural proteins and viral nucleic acid. Expression of genes encoding inflammatory cytokines and chemokines, including interleukin (IL)-1ß, IL-6, IL-15, tumour necrosis factor (TNF)-α, CXCL10, CCL8, interferon (IFN)-α, IFN-ß and IFN-γ, was higher in cats with FIP, consistent with inflammatory pathway activation. Expression of genes encoding transcription factors STAT1 and 2, regulating signalling pathways, particularly of the interferons, was also higher. Among cats without FIP, there were few differences between virus-positive and virus-negative MLNs; however, TLR9 and STAT2 expression were higher with infection, suggesting a direct viral effect. The study provides evidence for TLR involvement in the response to FCoV. This could open up new avenues for therapeutic approaches.

Prevalence and distribution of Borrelia and Babesia species in ticks feeding on dogs in the U.K.

Ticks were collected during March-July 2015 from dogs by veterinarians throughout the U.K. and used to estimate current prevalences and distributions of pathogens. DNA was extracted from 4750 ticks and subjected to polymerase chain reaction and sequence analysis to identify Borrelia burgdorferi sensu lato (Spirochaetales: Spirochaetaceae) and Babesia (Piroplasmida: Babesiidae) species. Of 4737 ticks [predominantly Ixodes ricinus Linneaus (Ixodida: Ixodidae)], B. burgdorferi s.l. was detected in 94 (2.0%). Four Borrelia genospecies were identified: Borrelia garinii (41.5%); Borrelia afzelli (31.9%); Borrelia burgdorferi sensu stricto (25.5%), and Borrelia spielmanii (1.1%). One Rhipicephalus sanguineus Latreille (Ixodida: Ixodidae), collected from a dog with a history of travel outside the U.K., was positive for B. garinii. Seventy ticks (1.5%) were positive for Babesia spp. Of these, 84.3% were positive for Babesia venatorum, 10.0% for Babesia vulpes sp. nov., 2.9% for Babesia divergens/Babesia capreoli and 1.4% for Babesia microti. One isolate of Babesia canis was detected in a Dermacentor reticulatus (Ixodida: Ixodidae) tick collected from a dog that had recently travelled to France. Prevalences of B. burgdorferi s.l. and Babesia spp. did not differ significantly between different regions of the U.K. The results map the widespread distribution of B. burgdorferi s.l. and Babesia spp. in ticks in the U.K. and highlight the potential for the introduction and establishment of exotic ticks and tick-borne pathogens.

High prevalence of Felis catus gammaherpesvirus 1 infection in haemoplasma-infected cats supports co-transmission.

Felis catus gammaherpesvirus 1 (FcaGHV1), a potential feline pathogen, has been identified in domestic cats from USA, Asia-Pacific and Central Europe. Transmission of FcaGHV1 during territorial encounters, a route not typical for gammaherpesviruses, is suggested by risk factor analyses from some regions. The aim of this study was to investigate the relationship between FcaGHV1 detection and risk factors, including haemoplasma co-infections, among UK cats to better understand transmission and global distribution of FcaGHV1. FcaGHV1 DNA was detected in blood samples from UK cats (11.56%; 95% confidence interval [CI], 7.47-16.84; n = 199). Logistic regression analyses showed that entire male cats were more likely to be FcaGHV1 positive than neutered male cats (odds ratio, 3.60; 95% CI, 1.22-10.46). Samples positive for DNA from any of three haemoplasma species had 19 times greater odds for testing positive for FcaGHV1 than haemoplasma negative cats in multivariable analyses after adjusting for age, sex and neuter status. Domestic cats in the UK can be infected with FcaGHV1, confirming that this virus is globally endemic. The identification of neuter status as a risk factor for FcaGHV1 detection provides further evidence to support transmission of this virus during territorial encounters and co-transmission with haemoplasmas is suggested.

Analysis of risk factors and prevalence of haemoplasma infection in dogs.

Mycoplasma haemocanis (Mhc) and 'Candidatus Mycoplasma haematoparvum' (CMhp) are canine haemoplasma species that can induce anaemia in immunocompromised and/or splenectomised dogs. This study aimed to determine the prevalence and phylogeny of canine haemoplasma species in dogs from Nigeria and describe any risk factors for infection. Canine haemoplasma species-specific and generic haemoplasma qPCR assays were used. The species-specific qPCR assays found Mhc infection in 18 of 245 dogs (7.3%), and CMhp infection in only one dog (0.4%). The generic haemoplasma qPCR assays were positive in 44 of 245 (17.9%) dogs. Twenty-five dogs had discordant qPCR results in that they were generic haemoplasma qPCR positive but species-specific qPCR negative. Further evaluation of these dogs by 16S rDNA sequencing gave limited results but 5 were confirmed to be infected with non-haemoplasma species: 2 Anaplasma phagocytophilum, 1 Anaplasma ovis, 1 Serratia marcescens and 1 Aerococcus spp. The 16S rRNA gene sequences from Mhc species showed>99.8% identity with each other and>99.6% identity with GenBank sequences, and resided in a single clade with other global Mhc and Mycoplasma haemofelis sequences, indicating low 16S rRNA genetic variability amongst this canine haemoplasma species.

Feline blood genotyping versus phenotyping, and detection of non-AB blood type incompatibilities in UK cats.

OBJECTIVES: The aim of this study was to determine the agreement between AB blood phenotyping and genotyping and determine whether non-AB blood type incompatibilities exist in UK cats. METHODS: Blood samples underwent phenotyping (A, B or AB) using microplate agglutination, and genotyping (AA, Ab or bb) using pyrosequencing of a fragment of the cytidine monophospho-N-acetylneuraminic acid hydroxylase gene. Non-AB blood type incompatibilities were investigated by cross-matching against reference blood of the same phenotype. RESULTS: Of 112 cats tested, 86 (77%) were blood phenotype A, 19 (17%) type B and 7 (6%) type AB. Genotype and initial phenotype agreed in 96% (107 of 112) of cats, but 5 were discordant; these were all B phenotype with either AA (n=2) or Ab (n=3) genotype. Two of the five cats had repeat blood samples tested: one was reclassified as phenotype A; the other remained phenotype B. Two cats had incompatibilities on minor cross-match, but these were attributed to phenotyping errors. CLINICAL SIGNIFICANCE: Unknown mutation(s) associated with phenotype B, resulting in false AA or Ab genotyping, were evident in a small number of cases in this study. No conclusive evidence for non-AB blood type incompatibilities was found.

Non-ribosomal phylogenetic exploration of Mollicute species: new insights into haemoplasma taxonomy.

Nine species of uncultivable haemoplasmas and several Mycoplasma species were examined by partial sequencing of two protein-encoding housekeeping genes. Partial glyceraldehyde-3-phosphate dehydrogenase (gapA) and heat shock protein 70 (dnaK) gene sequences were determined for these Mollicute species; in total nine gapA sequences and ten dnaK sequences were obtained. Phylogenetic analyses of these sequences, along with those of a broad selection of Mollicute species downloaded from GenBank, for the individual genes, and for the gapA and dnaK concatenated data set, revealed a clear separation of the haemoplasmas from other species within the Mycoplasma genus; indeed the haemoplasmas resided within a single clade which was phylogenetically detached from the pneumoniae group of Mycoplasmas. This is the first report to examine the use of gapA and dnaK, as well as a concatenated data set, for phylogenetic analysis of the haemoplasmas and other Mollicute species. These results demonstrate a distinct phylogenetic separation between the haemoplasmas and Mycoplasmas that corresponds with the biological differences observed in these species, indicating that further evaluation of the haemoplasmas' relationship with the Mycoplasma genus is required to determine whether reclassification of the haemoplasmas is necessary.

Cats with inflammatory bowel disease and intestinal small cell lymphoma have low serum concentrations of 25-hydroxyvitamin D.

BACKGROUND: Inflammatory bowel disease (IBD) and intestinal small cell lymphoma (ISCL) are common diseases in cats. The prevalence of alterations in the serum concentrations of fat soluble vitamins, such as vitamin D, in cats with IBD and ISCL is unknown. HYPOTHESIS/OBJECTIVES: The objective of this study was to measure serum 25 hydroxyvitamin D (25[OH]D) concentrations in cats with IBD or ISCL. Serum 25(OH)D also was measured in healthy cats, and in hospitalized ill cats with nongastrointestinal diseases. ANIMALS: Eighty-four cats were included in the study: 23 in the healthy group, 41 in the hospitalized ill group, and 20 in the IBD/ISCL group. METHODS: Retrospective study. Serum samples for vitamin D analysis were frozen at -20°C until serum 25(OH)D was measured by high-performance liquid chromatography (HPLC). RESULTS: Although there was overlap in serum 25(OH)D concentrations among the 3 groups, serum 25(OH)D concentrations were significantly lower in the cats with IBD or ISCL compared to healthy cats (P < .0001) and hospitalized ill cats (P = .014). In the IBD/ISCL group, there was a significant moderate positive correlation between serum albumin and 25(OH)D concentrations (r = 0.58, P = .018). CONCLUSION AND CLINICAL IMPORTANCE: The median serum concentration of 25(OH)D was significantly lower in cats with IBD/ISCL than in healthy cats and in hospitalized ill cats. Additional studies are required to elucidate the mechanism of hypovitaminosis D in cats with gastrointestinal diseases, to define the best management strategy to treat this complication, and to investigate its potential prognostic implications.

Infectious agent screening in canine blood donors in the United Kingdom.

OBJECTIVES: Transfusion of blood products is an important component of veterinary emergency medicine. Donors must be carefully selected to minimise risk of transmission of blood-borne infectious agents. This study was devised to assess the prevalence of such agents in healthy, non-travelled UK dogs screened as prospective donors. METHODS: Ethylenediaminetetraacetic acid blood samples from dogs donating blood between August 2007 and January 2012 were screened by polymerase chain reaction for haemotropic mycoplasmas, Bartonella, Babesia, Leishmania, Ehrlichia and Anaplasma spp. Dogs with positive or inconclusive results underwent repeat polymerase chain reaction testing. RESULTS: Four of 262 dogs had positive or inconclusive results at initial screening. Repeat polymerase chain reaction testing in each dog was negative, and none of the dogs developed clinical signs of disease. CLINICAL SIGNIFICANCE: The positive results on initial screening may have represented false positives from sample contamination or amplification of non-target DNA. It is also possible that dogs were infected at initial sampling but successfully cleared infection before repeat testing. The low number of positive results obtained suggests that prevalence of these agents in a population of healthy UK dogs is low and that use of blood products is unlikely to represent a significant risk of transmission of these diseases.

Haemoplasmas: lessons learnt from cats.

The haemotropic mycoplasmas (haemoplasmas) are a group of bacteria that can induce anaemia in a wide variety of mammals, including domestic cats and wild felids. Different feline haemoplasma species of varying pathogenicity exist, with the more pathogenic Mycoplasma haemofelis (Mhf) capable of inducing severe haemolytic anaemia, whilst 'Candidatus Mycoplasma haemominutum' (CMhm) and 'Candidatus Mycoplasma turicensis' (CMt) are infrequently associated with clinical disease. Chronic haemoplasma infections are common and cats are frequently infected by two or more haemoplasmas, complicating the clinical picture. The natural route of transmission of haemoplasma infection between cats has not yet been determined; however, experimental transmission has been demonstrated via both oral and parenteral administration of infected blood. To date the haemoplasmas have been unable to be cultured in vitro, and accurate diagnosis is currently reliant on detection of bacterial DNA using PCR assays. Treatment of clinical haemoplasmosis is focussed on supportive care in combination with empirical treatment with antimicrobials (tetracyclines or fluoroquinolones). A significant number of asymptomatic cats are positive for haemoplasma infection. These cats may play a role in the maintenance of haemoplasma infection within a population, and need to be considered when choosing potential blood donors. Use of PCR assays has provided an accurate method of diagnosing haemoplasma infection and quantifying response to therapy, including in non-feline host animals, as presumed zoonotic haemoplasma infections are now being documented. Recent advances in genome sequencing techniques have allowed the whole genome sequences of the feline haemoplasmas Mhf and CMhm to be derived, as well as a number of non-feline haemoplasma species. These data have aided the identification of antigens for use in the development of serological tests, allowed the proteomic study of haemoplasmas and provided clues as to how the haemoplasmas can persist within the host. Future areas of study include investigation of their zoonotic potential, mechanisms of immune system evasion and transmission of these emerging pathogens.

Phylogenetic analysis of feline coronavirus strains in an epizootic outbreak of feline infectious peritonitis.

BACKGROUND: Feline coronavirus (FCoV) infection is common. In a small percentage of cats, FCoV infection is associated with the fatal disease feline infectious peritonitis (FIP). Genetically distinct virulent and avirulent strains of FCoV might coexist within a cat population. OBJECTIVES: To determine whether the strains of FCoV in FIP-affected cats are closely related or genetically distinct from the fecally derived strains of FCoV in contemporary-asymptomatic cats during an epizootic outbreak of FIP. ANIMALS: Four cats euthanized because of FIP and 16 asymptomatic cats. METHODS: This prospective outbreak investigation was initiated during an outbreak of FIP in cats within or rehomed from a rescue/rehoming center. Postmortem samples were collected from cats with FIP and contemporaneous fecal samples from asymptomatic cats. RNA was purified from tissue and fecal samples, FCoV gene fragments were reverse transcribed, PCR-amplified using novel primers, and sequenced. Sequences were aligned with ClustalW and compared with published FCoV sequences. RESULTS: FCoV RNA was detected in all 4 FIP cat postmortem samples and in 9 of the 16 fecal samples from contemporary-asymptomatic cats. Novel primers successfully amplified fragments from 4 regions of the genome for all FCoV-positive samples. Phylogenetic analysis showed that the FIP-associated strains of FCoV from the outbreak were very closely related to the fecally derived strains of FCoV from contemporary-asymptomatic cats. CONCLUSIONS AND CLINICAL IMPORTANCE: Sequence analysis provided no evidence that genetically distinct virulent and avirulent strains of FCoV were present during this FIP outbreak.

Acute phase response to Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum' infection in FIV-infected and non-FIV-infected cats.

The pathogenicity of Haemoplasma spp. in cats varies with 'Candidatus Mycoplasma haemominutum' (CMhm) causing subclinical infection while Mycoplasma haemofelis (Mhf) often induces haemolytic anaemia. The aims of this study were to characterise the acute phase response (APR) of the cat to experimental infection with Mhf or CMhm, and to determine whether chronic feline immunodeficiency virus (FIV) infection influences this response. The acute phase proteins serum amyloid A (SAA), haptoglobin (Hp) and α-1-acid glycoprotein (AGP) concentrations were measured pre-infection and every 7-14 days up to day 100 post-infection (pi) in cats infected with either Mhf or CMhm. Half of each group of cats (6/12) were chronically and subclinically infected with FIV. Marbofloxacin treatment was given on days 16-44 pi to half of the Mhf-infected cats, and on days 49-77 pi to half of the CMhm-infected cats. FIV-infected animals had significantly lower AGP concentrations, and significantly greater Hp concentrations than non-FIV-infected cats when infected with CMhm and Mhf, respectively. Both CMhm and Mhf infection were associated with significant increases in SAA concentrations, while AGP concentrations were only significantly increased by Mhf infection. Mhf-infected cats had significantly greater SAA concentrations than CMhm-infected animals. Both Mhf and CMhm infections were associated with an APR, with Mhf infection inducing a greater response. Chronic FIV infection appeared to modify the APR, which varied with the infecting Haemoplasma species.

Real-time quantitative polymerase chain reaction detection of haemoplasmas in healthy and unhealthy dogs from Central Macedonia, Greece.

OBJECTIVES: The aims of this study were to determine the prevalence of canine haemoplasmas, Mycoplasma haemocanis and "Candidatus Mycoplasma haematoparvum" infection in Central Macedonia, Greece, and to evaluate any associations between canine haemoplasma infection and clinical presentation, selected laboratory data or the presence of ticks. METHODS: Genomic DNA was purified from excess blood (n=151) submitted for haematological examination. Purified DNA was subjected to species-specific quantitative polymerase chain reaction assays duplexed with a canine DNA control quantitative polymerase chain reaction. Clinical records were retrospectively examined and selected clinical parameters were compared to haemoplasma infection status. RESULTS: Nine samples were excluded due to inadequate canine DNA polymerase chain reaction results. Of the remaining 142 samples: eight (5·6%) were positive for M. haemocanis alone, six (4·2%) were positive for "Ca. M. haematoparvum" alone and one (0·7%) was dual positive. No association was found between haemoplasma status and age, sex, breed, health status, presence of anaemia, selected biochemistry parameters, presence of ectoparasites, routine ectoparasiticide treatment or the presence of selected tick-borne diseases.

Serum protein electrophoresis in 147 dogs.

Reference intervals for serum protein electrophoresis (SPE) were created from a group of 75 clinically healthy dogs and compared with SPE results obtained from clinical cases presented to the University of Bristol over an eight-and-a-half-year period. A total of 147 dogs, in which SPE had been performed, had complete case records available and thus met the inclusion criteria. Signalment and final diagnoses taken from the case records and SPE results were divided into normal and abnormal based on the newly established reference intervals. Cases were grouped according to the SPE protein fraction abnormalities and diagnosis using the DAMNITV classification system. Of the 147 cases, 140 (95.2 per cent) had abnormal SPE results. The most common protein fraction abnormality was decreased albumin (59.3 per cent) followed by a polyclonal increase in γ globulins (38.6 per cent). Decreased ß-1 globulins and increased ß-2 globulins were documented in 36.4 and 30.0 per cent of cases, respectively. The most common DAMNITV classification associated with abnormal SPE results was infectious/inflammatory disease, which was diagnosed in 79 of 140 cases (56.4 per cent). Monoclonal gammopathies were noted in eight dogs (5.7 per cent), and underlying lymphoproliferative disease was present in all cases where a diagnosis was achieved, including multiple myeloma (four dogs), splenic plasmacytoma (one dog), hepatic plasmacytoma (one dog) and lymphoma (one dog).

Haemoplasma infection is not a common cause of canine immune-mediated haemolytic anaemia in the UK.

OBJECTIVE: The aim of this study was to investigate whether the two canine haemoplasma species, Mycoplasma haemocanis and "Candidatus Mycoplasma haematoparvum," are commonly associated with immune-mediated haemolytic anaemia (IMHA) in UK dogs. METHODS: Three groups of dogs were recruited to the study: anaemic dogs with primary IMHA (n=37); anaemic dogs not meeting the inclusion criteria for primary IMHA (n=77) and non-anaemic dogs (n=113). DNA was extracted from 100 µl of blood and subjected to real-time quantitative polymerase chain reaction (qPCR) assays for both species of Mycoplasma. Each assay incorporated co-amplification of canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous internal control. RESULTS: Canine GAPDH was successfully amplified by qPCR from all 227 canine blood samples but none contained M. haemocanis or "Candidatus M. haematoparvum" DNA. CLINICAL SIGNIFICANCE: Haemoplasma infection is uncommon in dogs in the UK and no evidence was found that these organisms act as triggers for IMHA.

Coombs', haemoplasma and retrovirus testing in feline anaemia.

OBJECTIVE: To investigate the associations between Coombs' testing, haemoplasma and retroviral infections, and feline anaemia. METHODS: Haematology, Coombs' testing (including assessment of persistent autoagglutination) and selected infection testing (haemoplasma, feline leukaemia virus/feline immunodeficiency virus provirus) were performed in blood samples collected from 60 anaemic and 60 non-anaemic cats. RESULTS: No association between infection and anaemia or Coombs' positivity existed. Anaemic cats (21.7%) were significantly more likely than non-anaemic cats (0%) to have cold autoagglutination (P<0.0001), but significance (set at

Development and use of real-time PCR to detect and quantify Mycoplasma haemocanis and "Candidatus Mycoplasma haematoparvum" in dogs.

Two canine haemoplasma species have been recognised to date; Mycoplasma haemocanis (Mhc), which has been associated with anaemia in splenectomised or immunocompromised dogs, and "Candidatus Mycoplasma haematoparvum" (CMhp), recently described in an anaemic splenectomised dog undergoing chemotherapy. The study aim was to develop quantitative real-time PCR assays (qPCRs) incorporating an endogenous internal control to detect Mhc and CMhp and to apply these assays to DNA samples extracted from canine blood collected in Northern Tanzania (n=100) and from dogs presented to a Trinidadian veterinary hospital (n=185). QPCRs specific for Mhc and CMhp were designed using 16S rRNA gene sequence data, and each was duplexed with an assay specific for canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The assays detected < or =10 copies of a sequence-specific haemoplasma plasmid per reaction and neither assay showed cross-reactivity with 10(6) copies of the sequence-specific plasmid from the non-target canine haemoplasma species. Nineteen of the 100 Tanzanian samples (19%) were positive for Mhc alone and one (1%) was dually infected. One Trinidadian sample was negative for canine GAPDH DNA and was excluded from the study. Of the 184 remaining Trinidadian samples, nine (4.9%) were positive for Mhc alone, five (2.7%) for CMhp alone, and two (1.1%) dually infected. This is the first report of canine haemoplasma qPCR assays that use an internal control to confirm the presence of amplifiable sample DNA, and their application to prevalence studies. Mhc was the most commonly detected canine haemoplasma species.