Diagnosis of feline haemoplasma infection in Australian cats using a real-time PCR assay

Abstract

A total of 147 cats from the Sydney area of Australia that had blood samples submitted to veterinary laboratories were tested using a real-time polymerase chain reaction (PCR) assay able to detect and distinguish the two feline haemoplasma species. This sample number included two cats diagnosed with feline haemoplasma infection by routine blood smear examination. Statistical analysis was performed to evaluate associations between haemoplasma infection, age, sex, breed, haematocrit (HCT) values and anaemia status. One hundred and six cats (72.1%) were negative. Thirty-four cats (23.1%) were positive for ‘Candidatus M. haemominutum’, six cats (4.1%) were positive for M. haemofelis and one cat (0.7%) was positive for both species. Older, male, non-pedigree cats, with lower HCT values were more likely to be infected with ‘Candidatus M. haemominutum’. Significant inverse correlation was found between the amount of M. haemofelis DNA present in the blood and the HCT value. This report documents the existence of, and prevalence of, both haemoplasma species in a sample of cats in Australia and is the first to use quantitative real-time PCR in a prevalence study for haemoplasma infection.

Introduction

Based on phylogenetic analysis of 16S rRNA gene sequences, Haemobartonella felis has recently been reclassified within the genus Mycoplasma as Mycoplasma haemofelis (Neimark et al., 2001) and ‘Candidatus Mycoplasma haemominutum’ (Foley and Pedersen, 2001). Both species are collectively referred to as the feline haemoplasmas. Differences in pathogenicity exist between these species. Experimental infection with M. haemofelis often causes a severe haemolytic anaemia (Berent et al., 1998; Foley et al., 1998; Westfall et al., 2001). Conversely ‘Candidatus M. haemominutum’ infection does not usually induce severe anaemia (Foley et al., 1998; Westfall et al., 2001), although it has been suggested that co-infection with ‘Candidatus M. haemominutum’ and feline retroviruses (particularly FeLV) may result in significant anaemia (George et al., 2002).

Based on cytological evaluation, feline haemoplasma infection was first reported in Australia in 1961 (Manusu, 1961). Since this time sporadic cases have been reported (Harbutt, 1963; James and Brooks, 1993; Sheriff, 1974; Watson et al., 1978) and the prevalence of infection in different parts of Australia described by various authors has been low (Harbutt, 1969; Moore, 1986; Thomas and Robinson, 1994). Recent studies have shown that conventional PCR assays are more sensitive than cytology for the diagnosis of this infection (Berent et al., 1998; Foley et al., 1998; Westfall et al., 2001) and can distinguish between M. haemofelis and ‘Candidatus M. haemominutum’ infection. Conventional PCR has been successfully used to diagnose M. haemofelis infection in two cats from Perth (Clark et al., 2002).

Real-time PCR allows the detection of amplicon accumulation as it is synthesised using fluorogenic probes or intercalating dyes (Bustin, 2000). Post-amplification steps, such as gel electrophoresis, are not required. The assay is thus performed rapidly, and, since there is no need to open the reaction tubes following PCR, amplicon carry-over and false-positive results are far less likely than with conventional PCR. Importantly, the amount of fluorescence in real-time PCR is proportional to the amount of accumulated PCR product, so measurement of fluorescence during the exponential phase of PCR provides an accurate means to quantify DNA template in an unknown sample. Quantification of DNA template may be of particular importance with infectious agents such as the feline haemoplasmas which do not invariably cause clinical disease in the host. A real-time PCR assay for the detection and quantification of feline haemoplasma DNA has recently been described (Tasker et al., 2003b).

The aim of the current study was to evaluate the prevalence of infection with both M. haemofelis and ‘Candidatus M. haemominutum’ in blood samples collected from a convenience-sample of Australian cats using a real-time PCR assay. Statistical analysis was performed to document any associations between feline haemoplasma infection, patient characteristics and haematological values.

Materials and methods

Case material

Surplus EDTA-anticoagulated blood available (minimum 100 μl) from 55 feline samples submitted to the Diagnostic Services Laboratory, University Veterinary Centre, University of Sydney, Australia for routine haematological testing was stored at −20 °C. These samples were from cats with avariety of disease conditions and included two cats which had been diagnosed with feline haemoplasma infection based on blood smear examination. These blood samples were freeze-driedbefore transportation to the Department of Clinical Veterinary Science, University of Bristol, UK. Surplus EDTA-anticoagulated whole blood available from 92 feline samples collected at Paddington Cat Hospital, Sydney for haematological testing, was stored at −20 °C and then sent to the University of Bristol by courier service without refrigeration. Most of these samples were collected from non-healthy cats requiring haematological evaluation as part of a diagnostic profile, but eight samples from apparently healthy cats undergoing a pre-anaesthesia screen before neutering were also included. All samples used in this study were collected from cats in the Sydney area of Australia.

DNA extraction

On arrival in the UK, the freeze-dried blood samples were solubilised with 100 μl sterile phosphatebuffered saline before undergoing DNA extraction. Genomic DNA was prepared from 100 μl of the solubilised freeze-dried blood samples and the EDTA whole blood samples using the DNeasy 96 Tissue Kit (Qiagen, Crawley, UK) according to the manufacturer's instructions. For each plate of DNA extractions performed, three 100 μl aliquots of phosphate buffered saline underwent the DNA extraction protocol for subsequent PCR to screen for contamination during DNA extraction.

PCR amplification

All PCR amplifications were performed in 25 μl reaction volumes. A feline haemoplasma real-time PCR assay (Tasker et al., 2003b) was performed on all samples. Briefly, this assay comprised feline haemoplasma specific primers (Hf Forward 5′-ACGAAAGTCTGATGGAGCAATA-3′ and Hf Reverse 5′-ACGCCCAATAAATCCGRATAAT-3′) (Life Technologies, Paisley, Scotland) and Taqman probes specific for either M. haemofelis (hexachloro-fluorescein [HEX]-TACGAGGGATAATTATGATAGTACTTCGTGA-Black hole quencher [BHQ]1) or ‘Candidatus M. haemominutum’ (6-carboxyfluorescein [FAM]-AGCTTGATAGGAAATGATTAAGCCTTGA-BHQ1) (Cruachem Ltd, Glasgow, Scotland). PCR reactions comprised 12.5 μl 2×Platinum Quantitative PCR Supermix-UDG(Invitrogen Ltd, Glasgow, UK), 260 nM of each primer, 300 nM FAM Taqman probe, 200 nM HEX Taqman probe, 6 mM MgCl₂final concentration, 1 U additional Platinum Taq polymerase (2.2 U per 25 μl reaction), and 2 μl of template DNA, made up to a final volume of 25 μl with water. PCR was performed using an iCycler IQ system (Bio-Rad Laboratories Ltd, Hemel Hempstead, London) with an initial incubation at 50 °C for 3 minutes, then 95 °C for 2 minutes followed by 45 cycles of 95 °C for 5 seconds and 58 °C for 30 seconds. Fluorescence was detected at 530 nm and 570 nm at each annealing step. All samples were run in triplicate. DNA samples from known infected and non-infected cats and water were subjected to PCR as positive and negative controls. To confirm the presence of amplifiable DNA in the samples, and for standardisation across the samples, a real-time PCR for the detection of feline 28S rDNA was performed on all specimens. This assay comprised 12.5 μl 2×Qiagen Hotstart Enzyme (Qiagen), 200 nM of each primer (28S rDNA forward 5′-AGCAGGAGGTGTTGGAAGAG-3′ and 28S rDNA reverse 5′-AGGGAGAGCCTAAATCAAAGG-3′), 100 nM 28S rDNA Taqman probe (TEXAS RED-TGGCTTGTGGCAGCCAAGTGT-BHQ2), 4.5 mM MgCl₂final concentration and 2 μl of template DNA, made up to a final volume of 25 μl with water. PCR was performed using an iCycler IQ system (Bio-Rad Laboratories Ltd) with an initial incubation at 95 °C for 15 minutes followed by 45 cycles of 95 °C for 10 seconds and 60 °C for 15 seconds. Fluorescence was detected at 620 nm at each annealing step.

Statistical analysis

Statistical evaluation was carried out using Stata 7 (Stata Corporation, College Station, Texas, USA) and Statistics Package for Social Scientists for Windows Release 10.1.0 (SPSS, Woking, Surrey, UK). Descriptive statistics (including mean, median, standard deviation [SD] and standard error [SE]) were obtained for age, HCT and 28S rDNA threshold cycle (CT) values, and normality was tested for using the Kolmogorov–Smirnov Test. Cats were categorised as being anaemic or not based on HCT values where a value <0.25 L/L was taken to define anaemia.

Cats were divided into three groups based on the results of the real-time haemoplasma PCR assay: negative, ‘Candidatus M. haemominutum’ positive and M. haemofelis positive. The one cat which was positive for both species was not included in this aspect of statistical analysis. Using univariable analysis, these three groups were statistically analysed for differences in age, HCT, breed, sex, anaemic status and 28S rDNA CT values. Categorical variables were analysed using the chi-squared test, or Fisher's Exact test (when at least one expected value was less than five). Normally distributed data were analysed using one-way analysis of variance (ANOVA) and t-tests. To evaluate the quantitative aspect of the real-time PCR results, the HEX and FAM CT values for those cats positive for M. haemofelis and ‘Candidatus M. haemominutum’, respectively, were standardised to a given 28S rDNA CT value to allow comparison between cats. Scattergrams of the HCT values were plotted against the standardised HEX and FAM CT values. Bivariate correlation between these variables was measured by determination of Spearman's correlation coefficients.

For each of the predictor variables—age, sex, breed, HCT and anaemic status—and the outcome of ‘Candidatus M. haemominutum’ PCR positive status, unadjusted odds ratios and 95% confidence intervals were calculated, with categorisation of non-linear continuous variables, and analysis in contingency tables using the extended Mantel–Haenszel chi-squared test for trend. Stratification of data was performed to evaluate effect modification and confounding, and logistic regression multivariable analysis was performed to adjust for confounding using the outcome of ‘Candidatus M. haemominutum’ PCR positive status. This analysis was not possible for M. haemofelis PCR positive status due to the small number of infected cats identified. Predictor variables were selected for inclusion in the logistic regression model if the P value calculated in the univariable analysis was ≤0.20. Only cats with complete data sets for the variables examined were used in the logistic regression model. Variables were retained in the model if they resulted in a significant reduction in deviance of P<0.05 using a 2-tailed likelihood ratio chi-squared test (Hosmer and Lemeshow, 2000). Interactions identified by stratified analysis were tested in the model. A significant association was taken to be one with a P value ≤0.05.

Results

Haemoplasma real-time PCR results

All positive and negative control reactions were positive and negative, respectively. A total of 147 cats were tested for feline haemoplasma infection by real-time PCR. Of these 106 cats (72.1%) were negative, 34 cats (23.1%) were positive for ‘Candidatus M. haemominutum’, six cats (4.1%) were positive for M. haemofelis and one (0.7%) cat was positive for both species.

Univariable analysis of cats grouped by haemoplasma PCR status

Age

The age of 147 cats was known, ranging from 0.2 to 23.0 years (mean age 9.5 years, median 10.0 years, SD 5.5 years and SE 0.5 years). The dual infected cat was 6.0 years old. Those cats infected with ‘Candidatus M. haemominutum’ were significantly older (P=0.001) than cats negative for this organism. No significant differences in age were found between the M. haemofelis positive group and either the negative group or the ‘Candidatus M. haemominutum’ positive group.

HCT

The HCT values of 140 cats were known, ranging from 0.07 to 0.50 L/L (mean 0.34 L/L, median 0.35 L/L, SD 0.08 L/L and SE 0.007 L/L). The HCT value of the dual infected cat was 0.36 L/L. The HCT values of those cats infected with ‘Candidatus M. haemominutum’ (P=0.005) and M. haemofelis (P<0.001) were both significantly lower (Fig. 1) than the negative cats. Additionally the HCT values of those cats infected with M. haemofelis were also significantly lower than those cats infected with ‘Candidatus M. haemominutum’ (P=0.029).

Figure 1

HCT values of Australian cats grouped by haemoplasma infection status. Boxes represent the 25th, 50th (median) and 75th quartiles with whiskers extending to the greatest and smallest values. Black dots indicate outliers (cases with values greater than 1.5 box lengths from the upper or lower edge of the box). CMhm, ‘Candidatus M. haemominutum’ positive; Mhf, M. haemofelis positive. The HCT values of both the CMhm group (P=0.005) and the Mhf group (P<0.001) were significantly lower than the negative group. The HCT values of the Mhf group were also significantly lower than the CMhm group (P=0.029).

Breed

The breed status of 146 cats was known with 86 (58.9%) non-pedigree cats (79 domestic shorthairs, five domestic longhairs and two domestic mediumhairs) and 60 (41.8%) pedigree cats. The pedigree cats comprised 19 Burmese, eight Siamese/Orientals, seven Persians, three Chinchillas and one to two cats of miscellaneous other pedigree breeds. Interestingly, the ‘Candidatus M. haemominutum’ positive group contained a significantly greater number of non-pedigree cats than the negative group (P=0.001) (Fig. 2). No significant differences in breed distribution were found between the M. haemofelis positive group and either the negative group or the ‘Candidatus M. haemominutum’ positive group.

Figure 2

Breed distribution of Australian cats grouped by haemoplasma infection status. CMhm, ‘Candidatus M. haemominutum’ positive; Mhf, M. haemofelis positive. The CMhm group contained a significantly greater number of non-pedigree cats than the negative group (P=0.001).

Sex

The sex of all 147 cats was known, with 79 (53.7%) males and 68 (46.3%) females. The ‘Candidatus M. haemominutum’ positive group contained a greater number of male cats than the negative group although significance was not reached (P=0.074) (Fig. 3). No significant differences in sex distribution were found between the M. haemofelis positive group and either the negative group or the ‘Candidatus M. haemominutum’ positive group.

Figure 3

Sex distribution of Australian cats grouped by haemoplasma infection status. CMhm, ‘Candidatus M. haemominutum’ positive; Mhf, M. haemofelis positive. The CMhm group contained a greater number of male cats than the negative group, however, significance was not reached (P=0.074).

Anaemic status

The HCT of 140 cats was known, with 15 (10.7%) anaemic cats and 125 (89.3%) non-anaemic cats. No significant differences in the numbers of anaemic and non-anaemic cats were found between the cats grouped by haemoplasma status. The HCT values of the six cats infected with M. haemofelis only were 0.07, 0.09, 0.25, 0.26, 0.31 and 0.38 L/L.

28S rDNA CT values

All 147 samples were positive for 28S rDNA using the real-time PCR assay, and no significant difference in the 28S rDNA CT values was found between samples submitted as freeze-dried specimens and those submitted as whole blood. The 28S rDNA CT values ranged from 27.8 to 37.1 (mean 30.7, median 30.8, SD 1.49 and SE 0.12). No significant differences in the 28S rDNA CT values were found between the cats grouped by haemoplasma status.

Correlation between HCT values and haemoplasma CT values in infected cats

Scattergrams plotting HCT values and standardised HEX CT values of the cats positive for M. haemofelis only (six cats) and HCT values and standardisedFAM CT values of cats positive for ‘Candidatus M. haemominutum’ only (33 cats, since one cat did not have a HCT value available) showed some evidence of a positive correlation (Fig. 4). Bivariate analysis revealed a significant correlation between the HCT and HEX CT values of the M. haemofelis positive cats (Spearman r of 0.89, P=0.019) but no significant correlation was evident between the HCT and FAM CT values of the cats positive for ‘Candidatus M. haemominutum’ (Spearman r of 0.19, P=0.292).

Figure 4

Scattergrams showing adjusted HEX and FAM CT values and HCT values. The correlation coefficients (r) shown are those of Spearman.

Multivariable analysis for the outcome ‘Candidatus M. haemominutum’

Multivariable analysis was performed using data from the 132 cats with complete data sets. In a simple logistic regression model to estimate the probability of the outcome ‘Candidatus M. haemominutum’ with respect to the possible predictor variables, all three predictor variables (age, HCT and breed) which were significant in univariable analysis retained significance and sex, which had approached significance in univariable analysis, became significant (Tables 1 and 2). Older, male, non-pedigree cats, with lower HCT values, were thus at increased risk of being ‘Candidatus M. haemominutum’ positive. Stratified analysis revealed an interaction between HCT and sex, with an increase in HCT value being associated with a reduction in the likelihood of a cat being ‘Candidatus M. haemominutum’ positive in males but not females. Inclusion of this interaction term did not improve the fit of the logistic regression model.

Table 1

Unadjusted (crude) odds ratios for the predictor variables with P<0.2 and ‘Candidatus M. haemominutum’ PCR positive status

Variable	Odds ratio	95% confidence interval	χ²	P value
Age (years)	1.10^b	1.04–1.19	8.9^d	0.003
Sex
Female	1.0^a
Male	2.25	0.99–5.16	3.9	0.048
Breed
Pedigree	1.0^a
Non-pedigree	5.88	2.00–16.67	13.4	0.002
HCT (L/L)	1.92^c	1.37–2.70	13.9^d	0.056

Reference category.

Odds ratio for a one year unit increase in age.

Per unit decrease in HCT category where HCT values are divided into 4 categories of decreasing HCT based on quartile values.

Mantel–Haenszel extended chi-squared test for trend.

Table 2

Multivariable relationships between predictor variables and ‘Candidatus M. haemominutum’ PCR positive status, using logistic regression analysis (n=132)

	Regression coefficient	SE	Adjusted odds ratio	95% confidence interval	P value
Constant	1.00	1.17	2.7	0.38–19.30	0.393
Age (years)	0.09	0.05	1.1^a	1.00–1.20	0.045
Sex (male v female)	1.22	1.70	3.4	1.25–9.06	0.016
Breed (non-pedigree vs. pedigree)	−1.71	0.10	5.6	1.79–16.67	0.003
HCT (L/L)	−0.70	0.12	2.0^b	1.28–3.13	0.003

Odds ratio for a one year unit increase in age

Per unit decrease in HCT category where HCT values are divided into 4 categories of decreasing HCT based on quartile values.

Discussion

This is the first prevalence study to use real-time quantitative PCR for diagnosis of infection with both feline haemoplasma species. Additionally it is the first study in Australian cats to report the prevalence of both ‘Candidatus M. haemominutum’ and M. haemofelis in a convenience-sample of feline patients. The successful amplification of haemoplasma DNA in these specimens confirms the existence of both ‘Candidatus M. haemominutum’ and M. haemofelis in Australia. Indeed, sequencing of the full length of the 16S rRNA gene of a number of Australian haemoplasma isolates has confirmed the existence of species with near identical 16S rRNA sequences to those previously reported in the UK and the USA (Tasker et al., 2003c).

The current study was carried out using a convenience-sampled population, the limitations of which have been discussed previously (Sukuraet al., 1992). When assessing prevalence of infection, truly random samples should be used to generate data, but such samples are difficult to obtain in companion animal studies. Without doubt, differences exist in the sex, age and breed distributions of the cats sampled compared to the general Australian cat population. The cats studied also differ from cats in the general population because the majority were being investigated by veterinarians. This represents the most practical option of sampling and allows the study of a population of cats which can be influenced by veterinary intervention. The prevalence of haemoplasmainfection in this sample of Australian cats showed a similar pattern to that reported in the USA (Jensen et al., 2001) and the UK (Tasker et al., 2003a) in that ‘Candidatus M. haemominutum’ infection was common (23.1% cats in Australia v 12.7% in the USA and 16.9% in the UK), M. haemofelis infection was less common (4.1% cats in Australia v 4.5% in the USA and 1.4% in the UK) and dual infection was rare (0.7% cats in Australia v 2.3% in the USA and 0.2% in the UK).

Direct comparisons of the prevalences reported in these different studies are limited value because of likely differences in the populations sampled. Indeed, both the USA and current study were performed on samples received after general requests for blood samples from suspected haemoplasma-infected cats were made to local veterinarians. In the USA study, 37% of cats evaluated for haemoplasma infection by PCR were suspected of harbouring haemoplasmas based on the presence of anaemia, fever and/or cytological evidence of infection. The current Australian study included two cats which had been diagnosed with feline haemoplasma infection based on cytology, which were recruited following a request for haemoplasma-infected samples. These two cats were both M. haemofelis positive by real-time PCR and were both anaemic (HCT values of 0.07 and 0.09 L/L) and represent roughly a third of the total number of cats infected with M. haemofelis. Additionally, the Australian study included eight cats which were healthy at the time of sampling, all of which were negative for feline haemoplasma infection by PCR. Limitations therefore exist with extrapolation of results from this study to the general cat population of eastern Australia.

The type of PCR assay used for diagnosis could affect the prevalence results from the different countries. The USA and UK studies both used a conventional PCR assay although the forward and reverse primer sequences used were identical to those used in the real-time PCR assay here. Previously reported feline haemoplasma prevalence data from Australia (Moore, 1986; Thomas and Robinson, 1994) has been based on cytological diagnosis and found that between 0.6 and 2.4% of sick or anaemic cats were haemoplasma positive. The difference between this figure and those reported in the current study could be due to the increased sensitivity of PCR as a diagnostic test and/or the inclusion of two cats recruited due to their haemoplasma positive cytology.

The current study allowed the use of multivariable analysis to evaluate simultaneously risk factors for infection with ‘Candidatus M. haemominutum’. This was possible due to the large number of cats used in the study, allowing the logistic regression model to converge. It was found that older, male, non-pedigree cats, with lower HCT values, were significantly more likely to be infected with ‘Candidatus M. haemominutum’. In previous studies (which did not specify the haemoplasma species) a predisposition for haemoplasma infection in young cats has been suggested (Bedford, 1970; Flint et al., 1958; Grindem et al., 1990) although in two studies the risk for infection rose with increasing age, peaking at four to six years (Hayes and Priester, 1973) or seven to eight years (Nash and Bobade, 1986) before decreasing again. Since it appears that cats do not reliably eliminate the organism following infection (Berent et al., 1998; Foley et al., 1998; Westfall et al., 2001) the increasing odds ratio with age may merely reflect a cumulative risk of exposure to the organism with time. In previous studies conflicting reports on sex predisposition for infection have been given, with some studies suggesting no sex predisposition (Nash and Bobade, 1986; Yamaguchi et al., 1996) while others reporting males at an increased risk compared to females (Harrus et al., 2002; Hayes and Priester, 1973). Results of previous reports comparing pedigree and non-pedigree breeds have also been conflicting (Grindem et al., 1990; Hayes and Priester, 1973; Nash and Bobade, 1986). The UK study (Tasker et al., 2003a), which also used multivariable analysis to assess risk factors for infection with ‘Candidatus M. haemominutum’, found that, like the current study, older male non-pedigree cats were more likely to be ‘Candidatus M. haemominutum’ positive. The association between ‘Candidatus M. haemominutum’ infection and male non-pedigree cats may reflect exposure to routes of transmission or other risk factors not yet determined. These could include outdoor roaming, contact with vectors such as fleas or ticks or exposure to retrovirus infection. For example it is possible that feline immunodeficiency virus (FIV) is aconfounder for ‘Candidatus M. haemominutum’ infection, explaining the fact that older male non-pedigree cats are more likely to be infected with ‘Candidatus M. haemominutum’. Unfortunately the retrovirus status of the cats in the current study was not available so it is not possible to evaluate any possible associations between FIV and ‘Candidatus M. haemominutum’ infection.

It is interesting that ‘Candidatus M. haemominutum’ infected cats had significantly lower HCT values than the negative cats in this Australian study. In contrast, the UK study (Tasker et al., 2003a) did not find any statistical differences in haematological variables between ‘Candidatus M. haemominutum’ positive and negative cats. Although the HCT values of the ‘Candidatus M. haemominutum’ infected group were significantly lower than those of the negative cats in the current study, the mean±SD of the HCT values of the ‘Candidatus M. haemominutum’ infected group was 0.31±0.06 L/L, compared to 0.35±0.07 L/L in the negative group. Indeed only four of the 33 cats (12.1%) in the ‘Candidatus M. haemominutum’ infected group for which HCT values were available were considered to be anaemic (i.e. HCT <0.25 L/L). Thus, although ‘Candidatus M. haemominutum’ infection wasassociated with lower HCT values in the current study, it did not appear to be associated with overt anaemia in many infected cats. However, a recently published study (George et al., 2002) demonstrated a mild or moderate transient decrease in haemoglobin concentrations in seven of nine cats infected with ‘Candidatus M. haemominutum’, confirming that in certain situations ‘Candidatus M. haemominutum’ can result in anaemia. It is also possible that another variable not directly evaluated in this study is contributing to the association present between ‘Candidatus M. haemominutum’ and HCT values. FIV infection, for example, could be a confounder on this association in that FIV infection is itself associated with both ‘Candidatus M. haemominutum’ infection and anaemia, resulting in the apparent association between the latter two variables. Unfortunately, the unknown FIV status of the cats in the current study makes further investigation of this hypothesis impossible.

Cats infected with M. haemofelis also had significantly lower HCT values (mean±SD; 0.23±0.12 L/L) compared to the negative cats and interesting, also the ‘Candidatus M. haemominutum’ group. Again, and in contrast to the USA study (Jensenet al., 2001), no significant difference in the number of anaemic cats was found between the M. haemofelis infected group and the negative group, with only two of six cats (33.3%) classified as being anaemic. However, the number of M. haemofelis positive cats identified in this study was very small, and a larger number of such cats is likely to be required to confirm any statistical association with anaemia. M. haemofelis has certainly been shown to induce anaemia experimentally (Berentet al., 1998; Foley et al., 1998; Jensen et al., 2001; Westfall et al., 2001).

Positive PCR results (for both haemoplasma species) have been reported in asymptomatic cats (Tasker et al., 2003a), so the significanceof a positive PCR result should always be interpreted in light of observed physical findings and haematological features of the patient (Tasker and Lappin, 2002). Indeed, four of the six M. haemofelis only positive cats had HCT values of ≥ 0.25 L/L. Real-time PCR assays offer quantitative information on the amount of haemoplasma DNA present (Tasker et al., 2003b) in addition to confirming the presence of haemoplasma DNA. In this study it was hypothesised that those cases in which haemoplasma infection was thought to be the cause of the anaemia should have the greatest quantities of haemoplasma DNA present in blood. The correlation data enabled evaluation of the relationship between HCT values and the quantity of haemoplasma DNA present in blood samples to be assessed. Significant positive correlation was seen with M. haemofelis infected cats (Fig. 4) but not with the ‘Candidatus M. haemominutum’ infected cats. The significant positive correlation reported in the M. haemofelis infected cats occurred despite the inclusion of only a very small number of cats. Future studies should further evaluate the use of quantified haemoplasma DNA data in a larger and more representative cohort of cats since this may help with the interpretation of the significance of a positive PCR result, particularly with M. haemofelis. In particular, quantified data may help distinguish between chronic asymptomatic haemoplasma carrier cats and those with active infection.

Footnotes

Acknowledgements

Séverine Tasker was part-funded by the Royal College of Veterinary Surgeons Trust Fund West Scholarship for Feline Research. Richard Malik was supported by the Valentine Charlton Bequest of the Post Graduate Foundation of Veterinary Science of the University of Sydney.

References

Bedford

P.G.

Feline infectious anaemia in the London area, Veterinary Record 87, 1970, 305–310.

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 59, 1998, 1215–1220.

Bustin

S.A.

Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays, Journal of Molecular Endocrinology 25, 2000, 169–193.

Clark

Foster

S.F.

Spencer

P.B.

Detection of Haemobartonella felis (Candidatus Mycoplasma haemofelis) in Australia that is similar to the ‘Ohio’ strain, Australian Veterinary Journal 80, 2002, 703–704.

Flint

J.C.

Roepke

M.H.

Jensen

Feline infectious anaemia. I. Clinical aspects, American Journal of Veterinary Research 19, 1958, 164–168.

Foley

J.E.

Harrus

Poland

Chomel

Pedersen

N.C.

Molecular, clinical, and pathologic comparison of two distinct strains of Haemobartonella felis in domestic cats, American Journal of Veterinary Research 59, 1998, 1581–1588.

Foley

J.E.

Pedersen

N.C.

‘Candidatus Mycoplasma haemominutum’, a low-virulence epierythrocytic parasite of cats, International Journal of Systematic and Evolutionary Microbiology 51, 2001, 815–817.

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 63, 2002, 1172–1178.

Grindem

C.B.

Corbett

W.T.

Tomkins

M.T.

Risk factors for Haemobartonella felis infection in cats, Journal of the American Veterinary Medical Association 196, 1990, 96–99.

10.

Harbutt

P.R.

A clinical appraisal of feline infectious anaemia and its transmission under natural conditions, Australian Veterinary Journal 39, 1963, 401–404.

11.

Harbutt

P.R.

The incidence of Eperythrozoon (Haemobartonella) felis in Victoria, Australian Veterinary Journal 45, 1969, 87–88.

12.

Harrus

Klement

Aroch

Stein

Bark

Lavy

Mazaki-Tovi

Baneth

Retrospective study of 46 cases of feline haemobartonellosis in Israel and their relationships with FeLV and FIV infections, Veterinary Record 151, 2002, 82–85.

13.

Hayes

H.M.

Priester

W.A.

Feline infectious anaemia. Risk by age, sex and breed; prior disease; seasonal occurrence; mortality, Journal of Small Animal Practice 14, 1973, 797–804.

14.

Hosmer

D.W.

Lemeshow

Applied Logistic Regression, 2000, Wiley: New York.

15.

James

A.E.

Brooks

Salmonellosis and haemobartonellosis in kittens, Animal Technology 44, 1993, 233–237.

16.

Jensen

W.A.

Lappin

M.R.

Kamkar

Reagen

W.J.

Use of a polymerase chain reaction assay to detect and differentiate two strains of Haemobartonella felis infection in naturally infected cats, American Journal of Veterinary Research 62, 2001, 604–608.

17.

Manusu

H.P.

Infectious feline anaemia in Australia, Australian Veterinary Journal 37, 1961, 405.

18.

Moore

A.S.

A Survey of the Prevalence and Causes of Feline Anaemia in a Suburban Veterinary Practice, with Particular Reference to Haemobartonella felis, 1986, Faculty of Veterinary Science, University of Sydney: Sydney.

19.

Nash

A.S.

Bobade

P.A.

Haemobartonella felis infection in cats from the Glasgow area, Veterinary Record 119, 1986, 373–375.

20.

Neimark

Johansson

K.E.

Rikihisa

Tully

J.G.

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’, International Journal of Systematic and Evolutionary Microbiology 51, 2001, 891–899.

21.

Sheriff

Feline infectious anaemia, Veterinary Record 94, 1974, 385.

22.

Sukura

Grohn

Y.T.

Junttila

Palolahti

Association between feline immunodeficiency virus antibodies and host characteristics in Finnish cats, Acta Veterinaria Scandinavica 33, 1992, 325–334.

23.

Tasker

Binns

S.H.

Day

M.J.

Gruffydd-Jones

T.J.

Harbour

D.A.

Helps

C.R.

Jensen

W.A.

Olver

C.S.

Lappin

M.R.

Use of a PCR assay to assess prevalence and risk factors for Mycoplasma haemofelis and ‘Candidatus Mycoplasma haemominutum’ in cats in the United Kingdom, Veterinary Record 152, 2003a, 193–198.

24.

Tasker

Helps

C.R.

Day

M.J.

Gruffydd-Jones

T.J.

Harbour

D.A.

Use of Real-Time PCR to detect and quantify Mycoplasma haemofelis and ‘Candidatus Mycoplasma haemominutum’ DNA, Journal of Clinical Microbiology 41, 2003b, 439–441.

25.

Tasker

Helps

C.R.

Day

M.J.

Harbour

D.A.

Shaw

S.E.

Harrus

Baneth

Lobetti

R.G.

Malik

Beaufils

Belford

C.R.

Gruffydd-Jones

T.J.

Phylogenetic analysis of Haemplasma species—an international study, Journal of Clinical Microbiology 41, 2003c, 3877–3880.

26.

Tasker

Lappin

M.R.

Haemobartonella felis: recent developments in diagnosis and treatment, Journal of Feline Medicine and Surgery 4, 2002, 3–11.

27.

Thomas

J.B.

Robinson

W.F.

Anaemia in Western Australian cats: prevalence, patterns and causes, Australian Veterinary Practitioner 24, 1994, 210–217.

28.

Watson

A.D.J.

Farrow

B.R.H.

Hoskins

L.P.

Someobservations on treating cats infected with Haemobartonella felis , Australian Veterinary Practitioner 8, 1978, 129–132.

29.

Westfall

D.S.

Jensen

W.A.

Reagan

W.J.

Radecki

S.V.

Lappin

M.R.

Inoculation of two genotypes of Haemobartonella felis (California and Ohio variants) to induce infection in cats and the response to treatment with azithromycin, American Journal of Veterinary Research 62, 2001, 681–687.

30.

Yamaguchi

Macdonald

D.W.

Passanisi

W.C.

Harbour

D.A.

Hopper

C.D.

Parasite prevalence in free-ranging farm cats, Felis silvestris catus , Epidemiology & Infection 116, 1996, 217–223.