Molecular characterization and phylogenetic analysis of feline hemoplasmas in domestic cats in Iran

Document Type: Original Article

Authors

1 Department of Veterinary Internal Diseases, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

2 Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

Abstract

Three known feline hemoplasmas are Mycoplsama haemofelis, ‘Candidatus Mycoplasma haemominutum’ and ‘Candidatus Mycoplasma turicensis’. They are described as cause of feline infectious anemia in domestic and wild felids. Other blood parasites or blood-related pathogens like concurrent retroviral infections may deteriorate the clinical condition and severity of anemia. The aims of this study were molecular characterization and phylogenetic analysis of hemoplasmas in domestic cats in Iran for the first time. Blood samples were collected from 185 healthy and diseased domestic cats. Blood smears were prepared and hematological parameters were measured to determine possible anemia. Using 16S rRNA gene universal and species specific polymerase chain reactions with the following sequencing, 47 (25.40%) of cats were hemoplasma positive. Also, 17.02%, 72.50% and 40.40% of total positive samples were M. haemofelis, ‘Ca. M. haemominutum’ and ‘Ca.M. turicensis’ infected, respectively. 10 (21.20%) of hemoplasma positive cats had anemic blood profiles (HCT < 24.00%). All M. haemofelis infected cases were included. Partial 16S rRNA gene phylogenetic analysis revealed a high identity between the hemoplasma species found in this study and domestic cat sequences existing in GenBank. Phylogenetic analysis revealed 94.00% to 100% sequence identity between sequences of this study and existing sequences in Genbank. All hemoplasma isolates in this study were grouped within a single clade and additionally subdivided into two groups; haemofelis group including M. haemofelis and ‘Ca. M. turicensis’ and haemominutum group including ‘Ca. M. haemominutum’.

Keywords

Main Subjects

Introduction

 

Haemoplasmas are haemotropic mycoplasma bacteria in a very wide range of mammalians1 which are reclassified based on 16S rRNA gene from Rickettsia to the Mycoplasma genus. 2 Three feline haemoplasma species are described in domestic cats including M. haemofelis, Ca.M. haemominutum’ and ‘Ca.M. turicensis’1-4 causing hemolytic anemia in cats mostly in M. haemofelis infected cases.5,6Infected cats have no specific clinical signs typically in‘Ca. M. haemominutum’ and ‘Ca.M. turicensis’infection.7 Co-infection of haemoplasmas with some other pathogens like feline leukemia virus (FeLv) can lead to a severe and life threatening anemia.8,9

Since haemoplasmas could not be cultured10,11and cytological examinations of blood smears are not reliable, 12,13 other diagnostic methods mainly molecular assays are investigated.14-17 Using molecular techniques like polymerase chain reactions (PCR), detection, quantification and follow up of the treatment in hemotropic mycoplasmas are practicable.15,16 In addition, partial genome sequencing of common 16S rRNA gene in isolates from different hemoplasma species and complete genome sequencing project of M. haemofelis and Ca. Mycoplasma haemominutum,18-20facilitate studies about the evolution, pathogenesis and interspecies transmission in haemoplasmas. In a recent study from our group, the first report on the presence and clinical and hematological aspects of feline hemotropic mycoplasmas were described in domestic cats in Iran.12

The aim of this study was to investigate feline haemolplasma species in domestic cats with an approach to sequencing and phylogenetic analysis to determine the identity of detected isolates and compare to worldwide cat-derived isolates due to expansion of our knowledge about these hemotropic mycoplasmas.

 

Materials and Methods

 

Sample collection. EDTA-anticoagulated blood samples, collected from femoral vein into 2.5 mL tube (FL Medical S.r.l., Torreglia, Italy), were obtained from 185 healthy and diseased domestic cats (112 males and 73 females) of random ages, referred to three main referral diagnostic centers and an animal shelter between 2012 and 2014 in Tehran, Iran. Hematological parameters including white blood cell count, red blood cell count, hematocrit (HCT), hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin concentration and platelets count were measured using an automatic hemocytometer (Model Hema-screen 18; Hospitex diagnostic, Florence, Italy). Blood smears were prepared due to initial hemoplasma examination. Subsequently, blood samples were subjected to DNA extraction procedure for further molecular investigations.

DNA extraction. DNA was prepared of 100 µL blood sample using blood pathogens extraction kit (Molecular Biological System Transfer, Tehran, Iran) following the manufacturer's instructions and stored in – 20 ˚C prior to further investigations. For evaluating the extraction kit specificity and sensitivity, distilled water used as a negative control. The serial dilution of control positive samples (cloned DNA isolated from clinical cases, from the School of Veterinary Sciences, Bristol University, Bristol, UK and Bologna University, Bologna, Italy) with known copy number (down to 50 copy number) was extracted with the kit and subjected to the detecting conventional PCRs of feline haemolplasma species.

Diagnostic PCR assays. The control PCR to amplify a fragment of glyceraldehyde-3-phosphate dehydrogenase gene was applied to determine the quality of PCR procedure.21 Screening was performed based on previously described universal haemotropic mycoplasma conventional PCR detection method.22The positive samples with universal hemotropic mycoplasma PCR were subjected to the species specific conventional PCR of three feline haemolplasma species through formerly designed conventional PCR assays.23,24 Data are shown in Table 1.

 

Table 1. List of primers used in this study.

Species

Name

Primer sequence

Size of PCR product (bp)

Reference

Universal primers for hemotropic mycoplasma species

HBT-F

HBT-R

5'-ATACGGCCCATATTCCTACG-3'

5'-TGCTCCACCACTTGTTCA-3'

595 bp

8

Mycoplasma haemofelis

Candidatus Mycoplasma haemominutum

Jns-F

Jns-R

5'-ACGAAAGTCTGATGGAGCAATA-3'

5'-ACGCCCAATAAATCCG (A/G) ATAAT-3'

170 bp

193 bp

14

Candidatus Mycoplasma turicensis

Mt1-F

Mt2-R

5'-GTA TCC TCCATC AGA CAG AA-3'

5'-CGC TCC ATA TTT AAT TCCAA-3'

488 bp

22

GAPDH gene

GAPDH-F

GAPDH-R

5'-CCTTCATTGACCTCAACTACAT-3'

5'-CCAAAGTTGTCATGGATGACC-3'

277 bp

7

GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

 

Gene sequencing. A 595 bp fragment of the 16S rRNA gene, using universal haemotropic mycoplasma primers; 5´-ATACGGCCCATATTCCTACG-3´ and 5´-TGCTCCACCAC TTGTTCA-3´ as forward and reverse primers designed by Criado-Fornelio et al. was amplified22 and positive products were subjected to sequencing process using the sanger technique (ABI, 96-capillary XL).25

Statistical analysis. Statistical analysis was performed using SPSS software (version 16.0; IBM, New York, USA). Evaluation of normal distribution of hematological data was performed by a 1-sample Kolmogorov–Smirnov test. Data were analyzed with Fisher’s exact test and the independent t-tests and p < 0.05 is considered statistically significant. Sensitivity and specificity tests were performed with chi-square test. Sequence Data analysis and phylo-genic tree construction were performed with Genious (version 6.1.5; Biomatters Ltd., Auckland, New Zealand 2013). The evolutionary history was inferred using the Neighbor-Joining method.26[(IUCN),  #219;Felsenstein, 1985 #209] The optimal tree with the sum of branch length = 0.74248083 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.26 The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method27 and are in the units of the number of base substitutions per site. The analysis was involved 62 nucleotide sequences. All ambiguous positions were removed for each sequence pair. Evolutionary analyses were conducted in MEGA (version 6.0; Biodesign Institute, Tempe, USA).27

Results

 

From 185 samples, 47 (25.40%) were PCR-positive by universal haemotropic mycoplasma conventional PCR. The number of positive samples by species specific PCRs were M. haemofelis (n = 6), Candidatus M. haemominutum’ (n = 20), Candidatus M. turicensis’ (n = 5), M. haemofelis and ‘Candidatus M. turicensis’ (n = 2), and M. haemofelis and‘Candidatus M. turicensis’ (n = 14). There were co-infections of different feline hemotropic mycoplasmas.

From 47 hemoplasma positive samples, 10 (21.20%) had anemic hematological profiles (HCT < 24.00%) and 36 (76.50%) were male. All cats with only M. haemofelis infection or its co-infection with other species had anemic blood profiles. Overall anemia index factors including; hematocrit, red blood cells and hemoglobin in hemo-plasma positive samples are less than the same factors in hemoplasma negative cats. Data are shown in Tables 2, 3 and 4. Male cats were at higher risk of hemoplasma infection (p = 0.017, 95% confidence interval) with odds ratio of 2.699 greater than female infected cats.

Blood smears of 17 samples out of 185 total samples were positive for hemoplasmas (Fig. 1), of which five were negative with PCR. Using PCR as standard, cytology had a sensitivity of 28.57% and specificity of 96.50%.

 

Fig. 1. Wright-Giemsa stained cat blood smear at 100× with an oil immersion lens; hemoplasma bodies are shown with black arrows.

 

The 16S rRNA gene sequences derived from this study were submitted to Genbank with accession numbers of KX253960, KX253961, KX253962, KX253963, KX253964 for 16S rRNA genes of ‘Ca.M. haemominutum’ KX253965, KX253966 for M. haemofelis and KX253967 for ‘Ca.M. turicensis’ from domestic cats.28

Partial 16S rRNA gene sequence derived from the hemoplasma infected cats in the current study (accession number KX253960, KX253961, KX253962, KX253963, KX253964, KX253965, KX253966 and KX253967) showed high sequence identity to worldwide M. haemofelis, Ca.M. haemominutum’ and ‘Ca.M. turicensis’ sequences in Genbank.7,14,28,29 The KX253967 showed 97.16 to 100% sequence identity to ‘M. turicensis’ sequences. Sequences of Ca.M. haemominutum’ including KX253960, KX253961, KX253962, KX253963 and KX253964 ‘presented 94.12 to 100% identity to the reference sequence (accession NC 021007.1). M. haemofelis sequencesderived from this study,KX253965 and KX253966, showed 98.82 to 99.28% sequence identity to reference M. haemofelis sequence (NR 103953.1), (Fig. 2).

 

Table 2. Sex distribution in hemoplasma PCR-positive and -negative cats.

 

Male

Female

Total

Positive by smear examination

11

6

17

Negative by smear examination

101

67

168

Total

112

73

185

Positive by PCR

36

11

47

Negative by PCR

76

62

138

Total

112

73

185

 

 Table 3. Anemia distribution in three different hemoplasma species positive isolates.

 

Mhf

CMhm

CMt

Mhf-Cmt

CMhm-CMt

Total

Anemic profile

5

1

0

2

2

10

Not-anemic profile

0

15

5

0

17

37

Total

5

16

5

2

19

47

Mhf: Mycoplsama haemofelis, CMhm: Candidatus M. haemo-minutum, CMt: Candidatus M. turicensis, Mhf-Cmt: M. haemofelis and ‘Candidatus M. turicensis, CMhm-CMt: M. haemofelis and Candidatus M. turicensis.

 

 

Fig. 2. 595 bp fragment 16S rRNA gene phylogenetic analysis; the hemoplasma species found in this study and domestic cat sequences existing in GenBank. The following sequences are shown;‘Ca. M. haemominutum’ (cat, United Kingdom, AY150980; cat, USA, U88564; cat, China, AM745338; cat, United Kingdom, AF271154; cat, South Africa, AY150979; cat, United Kingdom, AY150981; cat, Hungary, EU128752; cat, Israel, AY150974; cat, Brazil, KM275257), ‘Candidatus M. turicensis’ (cat, Thailand, EU789559; cat, Brazil, EU442629; cat, Australia, DQ464425, cat, South Africa, DQ464424; cat, United Kingdom, DQ464420; cat, Brazil, KM275268; cat, Switzerland, DQ157150; cat, South Africa, DQ464418; cat, Iran, KJ530704), M. haemofelis (cat, USA, U95297; cat, USA, AF178677; cat, USA, AY069948; cat, France, AY150972; cat, Australia, AY150976; cat, Australia, AY150977; cat, United Kingdom, AY150948; cat, United Kingdom, AY150985; cat, Switzerland, DQ157160; cat, Thailand, EU145754; cat, USA, U88563; cat, South Africa, AF548631; cat, Japan, AY529632), Brucella abortus NR114469.

 

Table 4. Hematological parameters of hemoplasma positive and negative cats ⃰. Data are presented as Mean ± SD.

Parameter

Hemoplasma positive

Hemoplasma negative

Reference range30

Unit

Hematocrit

25.40

35.16

29.00 - 45.00

%

Hemoglobin

8.80

13.20

8.00 - 14.00

g dL-1

Red blood cells

6.40

8.54

6.00 - 10.00

106 µL-1

Mean corpuscular volume

50.00

47.60

41.00 - 54.00

fL

Mean corpuscular hemoglobin

16.10

15.32

13.30 - 17.50

pg

Mean corpuscular hemoglobin concentration

32.00

31.33

31.00 - 36.00

%

Platelets

1.80

3.70

2.30 - 6.80

105 µL-1

White blood cells

6.80

16.80

5.50 - 19.50

103 µL-1

Segmented neutrophil

2.13

9.56

2.50 - 12.50

103 µL-1

Band cell

0.05

0.21

0.00 - 0.30

103 µL-1

Lymphocyte

1.80

3.32

1.50 - 7.00

103 µL-1

Monocyte

0.05

0.07

0.00 - 0.85

103 µL-1

Eosinophil

0.04

0.20

0.00 - 1.50

103 µL-1

Basophil

0.00

0.00

Rare

103 µL-1

 ⃰ Age range of cats in this study was 3.34 ± 1.71 years old.

 

Discussion

 

This study was performed on domestic cats in Iran to investigate the molecular aspects of feline hemotropic mycoplasmas. The presence and co-infection of known feline hemoplasmas were shown by our group in another study in 2014. Moreover, it has been shown that sex, age and fighting history are predisposing risk factors of hemoplasma infection in cats.12 In agreement with previous studies, data obtained from the current study confirm that sex is a risk factor for hemoplasma infection.7,11,31,32

 Anemia (HCT < 24.00%) was detected in all M. haemofelis positive cats, either the infection was solely by M. haemofelis or combined with other hemoplasma species (totally seven out of ten anemic-hemoplasma positive cats). Data are shown in Table 3. There are several reports that the most pathogenic feline hemoplasma species is M. haemofelis.5,6 Some studies described that retrovirus infections could worsen the severity of the hemoplasma-induced anemia either in M. haemofelis infection or in anaemia following infection with less pathogenic hemoplasmas such as ‘Ca. M. haemominutum’ and ‘Ca. M. turicensis’.8,9 Unfortunately, serologically or molecularly screenings of retroviral co-infections were not possible in this study, which prohibited us from knowing whether co-infection might result in the hematological abnormalities found specially in low pathogen hemoplasma-induced infection.

There was no anemic case, infected only by ‘Ca. M. turicensis’, but some co-infected cats with ‘Ca. M. turicensis’ exhibited an anemic hematological profile. This result is in agreement with other studies shown the low pathogenicity of ‘Ca. M. turicensis’ infection solely.3, 33, 34

Smear examination is not a sensitive diagnostic tool, which traditionally is applied primarily in diagnostic labs to detect hemoplasmas.9,34 Comparing hemoplasma screening PCR results, as a described standard for hemo-tropic mycoplasmas detection, smear results in the current study confirm the same outcome with a sensitivity of 28.57% and specificity of 96.50% for cytology examination.

Co-infection of different feline hemoplasma species has been described in previous studies. In a study by Aquino et al., coinfection of two or three feline hemoplasma species was reported. M. haemofelis and‘Ca.M. haemominutum’ infection was the most frequent co-infection in the referred study. Meanwhile, ‘Ca.M. turicensis’ and ‘Ca.M. haemominutum’ co-infection was observed in the current study.35 In another study by Willi et al. in Switzerland, the association between ‘Ca.M. turicensis’ and ‘Ca.M. haemominutum’ has been shown which is in agreement with the results of present study. From 21 ‘Ca.M. turicensis’ positive samples, 14 samples (66.60%) were also ‘Ca.M. haemominutum’ positive.7

High sequence identity was observed between M. haemofelis, Ca.M. haemominutum’ and ‘Ca.M. turicensis’ isolates in this study and domestic cat derived sequences of three feline hemoplasmas in Genbank with no obvious geographical or host specificity grouping. Sequencing alignment with sequences derived from previous studies showed that worldwide isolated hemoplasmas are nearly identical irrespective of geographical or host origin.2,36,37

In uncultivable organisms such as hemoplasmas, phylogenetic analysis provides great information about their taxonomy. Several studies were investigated the phylogeny of hemoplasma on the basis of mainly two genes; 16S rRNA and RNase P RNA gene (rnpB) sequences.2,29,36-38

However, few studies have performed phylogenetic analysis on non-16S rRNA genes e.g., comparing the 16S rRNA gene, rnbp gene sequences have a higher nucleotide variation in closely related taxa.28,37-39

All hemoplasma isolates in this study were grouped within a single clade using 16S rRNA gene phylogenetic tree and were additionally subdivided into two groups; haemofelis group including two feline hemoplasma species, M. haemofelis and ‘Ca.M. turicensis’ and haemominutum group including ‘Ca.M. haemominutum’.

Hemoplasmas are not aggressive microorganisms with acute disease feature but could potentially cause anemia or deteriorate other infections like FeLV or FIV which could result in fatal anemia.12,16 To prevent clinical, diagnostic and therapeutic complications in pet clinics and having a greater health monitoring in cat populations, detection of subclinical and chronic infections like feline hemoplasmas could be very helpful.

Moreover, it should be considered that evolution relatedness and identity of these species in felids are so high and conserved with no obvious geographical or host specificity.

 

  1. Willi B, Boretti FS, Tasker S, et al. From haemobartonella to hemoplasma: Molecular methods provide new insights. Vet Microbiol 2007; 125(3-4): 197-209.
  2. Neimark H, Johansson KE, Rikihisa Y, et al. Proposal to transfer some members of the genera haemobartonella and eperythrozoon to the genus Mycoplasma with descriptions of 'Candidatus Mycoplasma haemofelis', 'Candidatus Mycoplasma haemomuris', 'Candidatus Mycoplasma haemosuis' and 'Candidatus Mycoplasma wenyonii'. Int J Syst Evol Microbiol 2001; 51(3): 891-899.
  3. Willi B, Boretti FS, Cattori V, et al. Identification, molecular characterization, and experimental transmission of a new hemoplasma isolate from a cat with hemolytic anemia in Switzerland. J Clin Microbiol 2005; 43(6): 2581-2585.
  4. Willi B, Museux K, Novacco M, et al. First morphological characterization of ‘Candidatus Mycoplasma turicensis’ using electron microscopy. Vet Microbiol 2011; 149(3-4): 367-373.
  5. Tasker S. Haemotropic mycoplasmas: What's their real significance in cats? J Feline Med Surg 2010; 12(5): 369-381.
  6. Berent LM, Messick JB, Cooper SK. Detection of Haemobartonella felis in cats with experimentally induced acute and chronic infections, using a polymerase chain reaction assay. Am J Vet Res 1998; 59(10): 1215-1220.
  7. Willi B, Boretti FS, Baumgartner C, et al. Prevalence, risk factor analysis, and follow-up of infections caused by three feline hemoplasma species in cats in Switzerland. J Clin Microbiol 2006; 44(3): 961-969.
  8. George JW, Rideout BA, Griffey SM, et al. Effect of preexisting FeLV infection or FeLV and feline immunodeficiency virus coinfection on pathogenicity of the small variant of Haemobartonella felis in cats. Am J Vet Res 2002; 63(8): 1172-1178.
  9. Macieira DB, De Menezes Rde C, Damico C, et al. Prevalence and risk factors for hemoplasmas in domestic cats naturally infected with feline immunodeficiency virus and/or feline leukemia virus in Rio de Janeiro-Brazil. J Feline Med Surg 2008; 10(2): 120-129.
  10. Foley JE, Pedersen NC. 'Candidatus Mycoplasma haemominutum', a low-virulence epierythrocytic parasite of cats. Int J Syst Evol Microbiol 2001; 51(Pt 3): 815-817.
  11. Gentilini F, Novacco M, Turba M, et al. Use of combined conventional and real-time PCR to determine the epidemiology of feline haemoplasma infections in northern Italy. J Feline Med Surg 2009; 11(4): 277-285.
  12. Ghazisaeedi F, Atyabi N, Zahrai Salehi T, et al. A molecular study of hemotropic mycoplasmas (hemoplasmas) in cats in Iran. Vet Clin Path 2014; 43(3): 381-386.
  13. Tasker S, Lappin MR. Haemobartonella felis:Recent developments in diagnosis and treatment. J Feline Med Surg 2002; 4(1): 3-11.
  14. Tasker S, Binns SH, Day MJ, et al. Use of real-time PCR to detect and quantify Mycoplasma haemofelis and "Candidatus Mycoplasma haemominutum" DNA. J Clin Microbiol 2003; 41(1): 439-441.
  15. Tasker S, Helps CR, Day MJ, et al. Use of a Taqman PCR to determine the response of Mycoplasma haemofelis infection to antibiotic treatment. J Microbiol Methods 2004; 56(1): 63-71.
  16. Wengi N, Willi B, Boretti FS, et al. Real-time PCR-based prevalence study, infection follow-up and molecular characterization of canine hemotropic mycoplasmas. Vet Microbiol 2008; 126(1-3): 132-141.
  17. Willi B, Meli ML, Luethy R, et al. Development and application of a universal hemoplasma screening assay based on the SYBR Green PCR principle. J Clin Microbiol 2009; 47(12): 4049-4054.
  18. Barker EN, Helps CR, Peters IR, et al. Complete genome sequence of Mycoplasma haemofelis, a hemotropic mycoplasma. J Bacteriol 2011; 193(8): 2060-2061.
  19. Messick JB, Santos AP, Guimaraes AMS. Complete genome sequences of two hemotropic mycoplasmas, Mycoplasma haemofelis strain Ohio2 and Mycoplasma suis strain Illinois. J Bacteriol 2011; 193(8):2068-2069.
  20. Barker EN, Darby AC, Helps CR, et al. Genome sequence for “Candidatus Mycoplasma haemominutum,” a low-pathogenicity hemoplasma species. J Bacteriol 2012; 194(4): 905-906.
  21. Birkenheuer AJ, Levy MG, Breitschwerdt EB. Development and evaluation of a seminested PCR for detection and differentiation of Babesia gibsoni (Asian Genotype) and B. canis DNA in canine blood samples. J Clin Microbiol 2003; 41(9): 4172-4177.
  22. Criado-Fornelio A, Martinez-Marcos A, Buling-Saraña A, et al. Presence of Mycoplasma haemofelis, Mycoplasma haemominutum and piroplasmids in cats from southern Europe: A molecular study. Vet Microbiol 2003; 93(4): 307-317.
  23. Jensen WA, Lappin MR, Kamkar S, et al. Use of a poly-merase chain reaction assay to detect and differentiate two strains of Haemobartonella felis in naturally infected cats. Am J Vet Res 2001; 62(4): 604-608.
  24. Santos AP, Messick JB, Biondo AW, et al. Design, optimization, and application of a conventional PCR assay with an internal control for detection of ‘Candidatus Mycoplasma turicensis’ 16S rDNA in domestic cats from Brazil. Vet Clin Path 2009; 38(4): 443-452.
  25. Pitulle C, Citron DM, Bochner B, et al. Novel bacterium isolated from a lung transplant patient with cystic fibrosis. J Clin Microbiol 1999; 37(12): 3851-3855.
  26. Felsenstein J. Confidence Limits on Phylogenies: An Approach Using the Bootstrap. Evol 1985; 39(4): 783-791.
  27. André MR, Adania CH, Allegretti SM, et al. Hemoplasmas in wild canids and felids in Brazil. J Zoo Wildl Med 2011; 42(2): 342-347.
  28. Willi B, Filoni C, Catão-Dias J, et al. Worldwide occurrence of feline hemoplasma infections in wild felid species. J Clin Microbiol 2007; 45(4): 1159-1166.
  29. Rikihisa Y, Kawahara M, Wen B, et al. Western immunoblot analysis of Haemobartonella muris and comparison of 16S rRNA gene sequences of H. muris, H. felis,and Eperythrozoon suis. J Clin Microbiol 1997; 35(4): 823-829.
  30. Weiss DJ, Wardrop KJ. Schalm's Veterinary hematology. 6th ed. New Jersey, USA: Wiley-Blackwell 2010; 813.
  31. Tasker S, Binns SH, Day M, et al. Use of a PCR assay to assess the prevalence and risk factors for Mycoplasma haemofelis and ‘Candidatus Mycoplasma haemominutum’ in cats in the United Kingdom. Vet Record 2003; 152(7): 193-198.
  32. Grindem CB, Corbett WT, Tomkins MT. Risk factors for Haemobartonella felis infection in cats. J Am Vet Med Assoc 1990; 196(1): 96-99.
  33. Willi B, Tasker S, Boretti FS, et al. Phylogenetic analysis of “Candidatus Mycoplasma turicensis” isolates from pet cats in the United Kingdom, Australia, and South Africa, with analysis of risk factors for infection. J Clin Microbiol 2006; 44(12): 4430-4435.
  34. Bauer N, Balzer HJ, Thüre S, et al. Prevalence of feline haemotropic mycoplasmas in convenience samples of cats in Germany. J Feline Med Surg 2008; 10(3): 252-258.
  35. Aquino LC, Hicks CA, Scalon MC, et al. Prevalence and phylogenetic analysis of haemoplasmas from cats infected with multiple species. J Microbiol Methods 2014; 107: 189-196.
  36. Tasker S, Helps CR, Belford CJ, et al. 16S rDNA comparison demonstrates near identity between an United Kingdom Haemobartonella felis strain and the American California strain. Vet Microbiol 2001; 81(1): 73-78.
  37. Tasker S, Helps CR, Day MJ, et al. Phylogenetic analysis of hemoplasma species: an international study. J Clin Microbiol 2003; 41(8): 3877-3880.
  38. Peters IR, Helps CR, McAuliffe L, et al. RNase P RNA gene (rnpb) phylogeny of hemoplasmas and other mycoplasma species. J Clin Microbiol 2008; 46(5): 1873-1877.
  39. Peters, IR, Helps CR, Willi B, et al. The prevalence of three species of feline haemoplasmas in samples submitted to a diagnostics service as determined by three novel real-time duplex PCR assays. Vet Microbiol 2008; 126(1-3): 142-150.