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1.  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▿  
Journal of Clinical Microbiology  2006;44(12):4430-4435.
Two hemotropic mycoplasmas have been recognized in cats, Mycoplasma haemofelis and “Candidatus Mycoplasma haemominutum.” We recently described a third feline hemoplasma species, designated “Candidatus Mycoplasma turicensis,” in a Swiss cat with hemolytic anemia. This isolate induced anemia after experimental transmission to two specific-pathogen-free cats and analysis of the 16S rRNA gene revealed its close relationship to rodent hemotropic mycoplasmas. The agent was recently shown to be prevalent in Swiss pet cats. We sought to investigate the presence and clinical importance of “Candidatus Mycoplasma turicensis” infection in pet cats outside of Switzerland and to perform the molecular characterization of isolates from different countries. A “Candidatus Mycoplasma turicensis”-specific real-time PCR assay was applied to blood samples from 426 United Kingdom (UK), 147 Australian, and 69 South African pet cats. The 16S rRNA genes of isolates from different countries were sequenced and signalment and laboratory data for the cats were evaluated for associations with “Candidatus Mycoplasma turicensis” infection. Infections were detected in samples from UK, Australian, and South African pet cats. Infection was associated with the male gender, and “Candidatus Mycoplasma haemominutum” and M. haemofelis coinfection. Coinfected cats exhibited significantly lower packed cell volume (PCV) values than uninfected cats. Phylogenetic analyses revealed that some Australian and South African “Candidatus Mycoplasma turicensis” isolates branched away from the remaining isolates. In summary, “Candidatus Mycoplasma turicensis” infection in pet cats exists over a wide geographical area and significantly decreased PCV values are observed in cats coinfected with other feline hemoplasmas.
doi:10.1128/JCM.00987-06
PMCID: PMC1698426  PMID: 17035497
2.  Prevalence, Risk Factor Analysis, and Follow-Up of Infections Caused by Three Feline Hemoplasma Species in Cats in Switzerland 
Journal of Clinical Microbiology  2006;44(3):961-969.
Recently, a third novel feline hemotropic Mycoplasma sp. (aka hemoplasma), “Candidatus Mycoplasma turicensis,” in a cat with hemolytic anemia has been described. This is the first study to investigate the prevalence, clinical manifestations, and risk factors for all three feline hemoplasma infections in a sample of 713 healthy and ill Swiss cats using newly designed quantitative real-time PCR assays. “Candidatus Mycoplasma haemominutum” infection was detected in 7.0% and 8.7% and Mycoplasma haemofelis was detected in 2.3% and 0.2% of healthy and ill cats, respectively. “Candidatus Mycoplasma turicensis” was only detected in six ill cats (1.1%); three of them were coinfected with “Candidatus Mycoplasma haemominutum.” The 16S rRNA gene sequence of 12 Swiss hemoplasma isolates revealed >98% similarity with previously published sequences. Hemoplasma infection was associated with male gender, outdoor access, and old age but not with retrovirus infection and was more frequent in certain areas of Switzerland. “Candidatus Mycoplasma haemominutum”-infected ill cats were more frequently diagnosed with renal insufficiency and exhibited higher renal blood parameters than uninfected ill cats. No correlation between hemoplasma load and packed cell volume was found, although several hemoplasma-infected cats, some coinfected with feline immunodeficiency virus or feline leukemia virus, showed hemolytic anemia. High M. haemofelis loads (>9 × 105 copies/ml blood) seem to lead to anemia in acutely infected cats but not in recovered long-term carriers. A repeated evaluation of 17 cats documented that the infection was acquired in one case by blood transfusion and that there were important differences among species regarding whether or not antibiotic administration led to the resolution of bacteremia.
doi:10.1128/JCM.44.3.961-969.2006
PMCID: PMC1393118  PMID: 16517884
3.  Identification, Characterization, and Application of a Recombinant Antigen for the Serological Investigation of Feline Hemotropic Mycoplasma Infections ▿ ‡  
Clinical and Vaccine Immunology : CVI  2010;17(12):1917-1925.
In felids, three hemotropic mycoplasma species (hemoplasmas) have been described: Mycoplasma haemofelis, “Candidatus Mycoplasma haemominutum,” and “Candidatus Mycoplasma turicensis.” In particular, M. haemofelis may cause severe, potentially life-threatening hemolytic anemia. No routine serological assays for feline hemoplasma infections are available. Thus, the goal of our project was to identify and characterize an M. haemofelis antigen (DnaK) that subsequently could be applied as a recombinant antigen in a serological assay. The gene sequence of this protein was determined using consensus primers and blood samples from two naturally M. haemofelis-infected Swiss pet cats, an experimentally M. haemofelis-infected specific-pathogen-free cat, and a naturally M. haemofelis-infected Iberian lynx (Lynx pardinus). The M. haemofelis DnaK gene sequence showed the highest identity to an analogous protein of a porcine hemoplasma (72%). M. haemofelis DnaK was expressed recombinantly in an Escherichia coli DnaK knockout strain and purified using Ni affinity, size-exclusion, and anion-exchange chromatography. It then was biochemically and functionally characterized and showed characteristics typical for DnaKs (secondary structure profile, thermal denaturation, ATPase activity, and DnaK complementation). Moreover, its immunogenicity was assessed using serum samples from experimentally hemoplasma-infected cats. In Western blotting or enzyme-linked immunosorbent assays, it was recognized by sera from cats infected with M. haemofelis, “Ca. Mycoplasma haemominutum,” and “Ca. Mycoplasma turicensis,” respectively, but not from uninfected cats. This is the first description of a full-length purified recombinant feline hemoplasma antigen that can readily be applied in future pathogenesis studies and may have potential for application in a diagnostic serological test.
doi:10.1128/CVI.00282-10
PMCID: PMC3008192  PMID: 20876820
4.  Detection of Humoral Response Using a Recombinant Heat Shock Protein 70, DnaK, of Mycoplasma haemofelis in Experimentally and Naturally Hemoplasma-Infected Cats▿ †  
Clinical and Vaccine Immunology : CVI  2010;17(12):1926-1932.
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.
doi:10.1128/CVI.00320-10
PMCID: PMC3008206  PMID: 20926695
5.  Identification, Molecular Characterization, and Experimental Transmission of a New Hemoplasma Isolate from a Cat with Hemolytic Anemia in Switzerland 
Journal of Clinical Microbiology  2005;43(6):2581-2585.
Recently, there has been a growing interest in hemotropic mycoplasmal species (also known as the hemoplasmas), the causative agents of infectious anemia in several mammalian species. In felids, two different hemoplasma species have been recognized: Mycoplasma haemofelis (formerly Haemobartonella felis) and “Candidatus Mycoplasma haemominutum.” Recently developed molecular methods have allowed sensitive and specific identification and quantification of these agents in feline blood samples. In applying these methods to an epidemiological study surveying the Swiss pet cat population for hemoplasma infection, we discovered a third novel and unique feline hemoplasma isolate in a blood sample collected from a cat that had exhibited clinical signs of severe hemolytic anemia. This agent was readily transmitted via intravenous inoculation to two specific-pathogen-free cats. One of these cats was immunocompromised by the administration of methylprednisolone acetate prior to inoculation, and this cat developed severe anemia. The other immunocompetent cat showed a moderate decrease in packed cell volume. Additionally, an increase in red blood cell osmotic fragility was observed. Sequencing of the entire 16S rRNA gene of the new hemoplasma isolate and phylogenetic analysis showed that the isolate was most closely related to two rodent hemotropic mycoplasmal species, M. coccoides and M. haemomuris. A quantitative real-time PCR assay specific for this newly discovered agent was developed, which will be a prerequisite for the diagnosis of infections with the new hemoplasma isolate.
doi:10.1128/JCM.43.6.2581-2585.2005
PMCID: PMC1151947  PMID: 15956367
6.  Hemotropic mycoplasma prevalence in shelter and client-owned cats in Saskatchewan and a comparison of polymerase chain reaction (PCR) — Results from two independent laboratories 
The primary objective of this study was to determine the prevalence of subclinical hemotropic mycoplasma (HM) infections in 2 distinct feline populations: cats from a local shelter and client-owned cats presented for elective procedures (vaccination, ovariohysterectomy, orchiectomy) at the Western College of Veterinary Medicine — Veterinary Teaching Hospital (WCVM-VTH). The second objective of this study was to evaluate the inter-test agreement of 2 independent conventional polymerase chain reaction (PCR) assays used for the diagnosis of feline HM-infections.
Fifty-eight clinically healthy shelter cats and 57 clinically healthy client-owned cats were screened for subclinical HM-infection using a conventional PCR assay to detect the 16S rRNA of Mycoplasma haemofelis and “Candidatus M. haemominutum.” All cats in both groups had normal physical examinations. Sex, age (estimated for shelter cats), breed, reproductive status and the presence or absence of ectoparasites were determined. Packed cell volume (PCV), total protein, retroviral status, and blood smear evidence of HM-infection were evaluated. Subclinical HM-infection was identified by PCR assay in 12% (7/58) of the shelter cats and 4% (2/57) of the client-owned cats. M. haemofelis was found in 3/7 HM-infected shelter cats and 2/2 of the HM-infected client-owned cats; “Candidatus M. haemominutum” was found in 4/7 of the HM-infected shelter cats. There was no significant difference in prevalence of HM-infection between the populations (OR 3.8, 95% CI 0.75 to 19, P = 0.16), and no risk factors for infection were identified in either population.
Blood samples from 44 cats with known PCR results (26 cats sampled in the prevalence study and 18 clinical cases) were submitted to a second independent laboratory for HM PCR assay to assess inter-laboratory agreement. There was substantial, but not complete agreement between the 2 independent laboratories for PCR detection of M. haemofelis (κ = 0.66) and “Candidatus M. haemominutum” (κ = 0.70).
PMCID: PMC2851730  PMID: 20592837
7.  In vivo transmission studies of ‘Candidatus Mycoplasma turicensis’ in the domestic cat 
Veterinary Research  2009;40(5):45.
The natural transmission routes of the three feline haemotropic mycoplasmas – Mycoplasma haemofelis, ‘Candidatus Mycoplasma haemominutum’, and ‘Candidatus Mycoplasma turicensis’ (CMt) – are largely unknown. Since CMt has been detected in the saliva of infected cats using PCR, we hypothesised that direct transmission via social or aggressive contact may occur. The aim of this study was to evaluate this transmission route. CMt-positive saliva and blood samples were obtained from three prednisolone-treated specific pathogen-free (SPF) cats that were infected intraperitoneally with CMt. Five SPF cats were inoculated with CMt-positive saliva or blood subcutaneously to mimic cat bites, and five cats were inoculated orally with blood or oronasally with saliva to mimic social contact. Blood samples were monitored for CMt infection using quantitative real-time PCR and for seroconversion using a novel western blot assay. Neither oronasal nor subcutaneous inoculation with CMt-positive saliva led to CMt infection in the recipient cats, as determined by PCR, independent of prior prednisolone treatment. However, when blood containing the same CMt dose was given subcutaneously, 4 of the 5 cats became PCR-positive, while none of the 5 cats inoculated orally with up to 500 μL of CMt-positive blood became PCR-positive. Subsequently, the latter cats were successfully subcutaneously infected with blood. All 13 CMt-exposed cats seroconverted. In conclusion, CMt transmission by social contact seems less likely than transmission by aggressive interaction. The latter transmission may occur if the recipient cat is exposed to blood from an infected cat.
doi:10.1051/vetres/2009028
PMCID: PMC2701178  PMID: 19505421
haemotropic mycoplasma; transmission; ‘Candidatus Mycoplasma turicensis’; real-time TaqMan PCR; seroconversion
8.  Haemotropic mycoplasmas 
Practical relevance
The feline haemotropic mycoplasmas (‘haemoplasmas') are a group of bacteria that can induce haemolytic anaemia in cats. Mycoplasma haemofelis is the most pathogenic of the species; ‘Candidatus Mycoplasma haemominutum’ and ‘Candidatus Mycoplasma turicensis’ are less pathogenic. The natural route of transmission of feline haemoplasma infection has not been confirmed, but fleas are implicated. When disease results, common clinical signs are pallor, lethargy, anorexia, weight loss, depression, dehydration and pyrexia. Treatment with tetracyclines or fluoroquinolones is usually effective at resolving clinical disease, but clearance of infection may not result.
Global importance
The feline haemoplasmas are found worldwide, although prevalence varies geographically.
Patient group
Older male non-pedigree cats are believed to be at increased risk of haemoplasma infection, although younger cats are possibly more likely to show clinical disease associated with M haemofelis.
Clinical challenges
The significance of feline haemoplasma infection is difficult to determine due to the existence of asymptomatic carrier cats and the variable pathogenicity of the haemoplasma species. Polymerase chain reaction (PCR) results should be interpreted in the light of the patient's clinical signs and haematological findings, infecting haemoplasma species and level of haemoplasma DNA present in the blood. Trial antibiotic treatment for haemoplasmosis may be warranted in suspected cases while awaiting PCR results.
Evidence base
Aspects of feline haemoplasmosis, particularly risk factors, pathogenesis, diagnostic methods and treatment, have been the focus of much recent research. This article draws on the current evidence base with a view to helping clinicians diagnose and manage cases more effectively.
doi:10.1016/j.jfms.2010.03.011
PMCID: PMC2880789  PMID: 20417898
9.  Survey of Toxoplasma gondii and Neospora caninum, haemotropic mycoplasmas and other arthropod-borne pathogens in cats from Albania 
Parasites & Vectors  2014;7:62.
Background
Albania is a country on the western part of the Balkan Peninsula. The Mediterranean climate is favourable for the stable development of many arthropod species, which are incriminated as vectors for various agents. Recently, several papers have reported on epidemiological aspects of parasitic diseases including vector-borne disease agents of dogs with zoonotic characteristics in Albania. However, data on the epidemiology of feline parasitic and bacterial agents in Albania is scarce.
Methods
Serum and EDTA-blood samples collected from 146 domestic cats from Tirana during 2008 through 2010 were examined for exposure to Toxoplasma gondii, Neospora caninum, Leishmania infantum, and Anaplasma spp. with IFAT, for infection with L. infantum, A. phagocytophilum, Bartonella spp. and haemotropic mycoplasmas with conventional PCR and real-time PCR and for Dirofilaria immitis with antigen ELISA. Additionally blood smear microscopy was carried out for detection of blood-borne pathogens.
Results
Antibodies to T. gondii (titre ≥1:100) were demonstrated in 91 cats (62.3%). Antibodies to N. caninum (titre ≥1:100), L. infantum (titre ≥1:64) and Anaplasma spp. (titre ≥1:100) were found in the serum of 15 (10.3%), 1 (0.7%) or 3 (2.1%) cats, respectively. DNA of haemotropic mycoplasmas was detected in the blood of 45 cats (30.8%), namely Candidatus Mycoplasma haemominutum (21.9%), Mycoplasma haemofelis (10.3%), and Candidatus Mycoplasma turicensis (5.5%), with ten cats harbouring co-infections of two mycoplasmas each; blood from one cat was PCR positive for Bartonella henselae. No DNA of Leishmania spp. and A. phagocytophilum or circulating D. immitis antigen was detected in any cat sample. The overall prevalence of haemotropic mycoplasmas was significantly higher in male compared to female cats (40.6% vs. 24.1%, p = 0.0444); and age was associated positively with the prevalence of antibodies to T. gondii (p = 0.0008) and the percentage of haemotropic mycoplasma infection (p = 0.0454).
Conclusions
With the broad screening panel including direct and indirect methods applied in the present study, a wide spectrum of exposure to or infection with parasitic or bacterial agents was detected.
doi:10.1186/1756-3305-7-62
PMCID: PMC3926860  PMID: 24517118
Cat; Albania; Toxoplasma gondii; Neospora caninum; Leishmania infantum; Anaplasma spp.; Bartonella henselae; haemotropic mycoplasmas
10.  Hemotropic mycoplasmas in little brown bats (Myotis lucifugus) 
Parasites & Vectors  2014;7:117.
Background
Hemotropic mycoplasmas are epicellular erythrocytic bacteria that can cause infectious anemia in some mammalian species. Worldwide, hemotropic mycoplasmas are emerging or re-emerging zoonotic pathogens potentially causing serious and significant health problems in wildlife. The objective of this study was to determine the molecular prevalence of hemotropic Mycoplasma species in little brown bats (Myotis lucifugus) with and without Pseudogymnoascus (Geomyces) destrucans, the causative agent of white nose syndrome (WNS) that causes significant mortality events in bats.
Methods
In order to establish the prevalence of hemotropic Mycoplasma species in a population of 68 little brown bats (Myotis lucifugus) with (n = 53) and without (n = 15) white-nose syndrome (WNS), PCR was performed targeting the 16S rRNA gene.
Results
The overall prevalence of hemotropic Mycoplasmas in bats was 47%, with similar (p = 0.5725) prevalence between bats with WNS (49%) and without WNS (40%). 16S rDNA sequence analysis (~1,200 bp) supports the presence of a novel hemotropic Mycoplasma species with 91.75% sequence homology with Mycoplasma haemomuris. No differences were found in gene sequences generated from WNS and non-WNS animals.
Conclusions
Gene sequences generated from WNS and non-WNS animals suggest that little brown bats could serve as a natural reservoir for this potentially novel Mycoplasma species. Currently, there is minimal information about the prevalence, host-specificity, or the route of transmission of hemotropic Mycoplasma spp. among bats. Finally, the potential role of hemotropic Mycoplasma spp. as co-factors in the development of disease manifestations in bats, including WNS in Myotis lucifugus, remains to be elucidated.
doi:10.1186/1756-3305-7-117
PMCID: PMC3994326  PMID: 24655520
Hemotropic mycoplasma; Bat; Haemoplasma; Mycplasma; WNS
11.  A comparison of real-time PCR and reverse line blot hybridization in detecting feline haemoplasmas of domestic cats and an analysis of risk factors associated with haemoplasma infections 
Background
Three species of feline haemoplasma are recognised: Mycoplasma haemofelis (Mhf), ‘Candidatus Mycoplasma haemominutum’ (CMhm) and ‘Candidatus Mycoplasma turicensis (CMt). This study compared a reverse line blot hybridization (RLB) assay for simultaneous detection of Mhf, CMhm with three separate quantitative real-time polymerase chain reaction (qPCR) assays used for diagnosis of Mhf, CMhm and CMt. The RLB and qPCR assays were applied to DNA extracted from blood samples collected from 154 cats from Trinidad and Tobago.
Results
CMhm and Mhf DNA were detected using both RLB and qPCR. CMt DNA was detected by qPCR only. Comparing RLB and qPCR for the detection of CMhm DNA, 40 (26.3%) and 48 (31.6%) cats, respectively, were positive. The difference was more marked for Mhf, with RLB detecting a total of only 11 (7.2%) positive cats whereas qPCR detected 41 (27.0%) positive cats. Using qPCR as a gold standard, haemoplasma infected cats were more likely to be retrovirus positive (OR = 5.68, P = 0.02) and older (median age 5.5 years), than non-infected cats. In addition, CMhm positive cats were more likely to be male (OR = 3.4, P = 0.04).
Conclusions
Overall the qPCR was more sensitive than RLB. In addition, age (median 5.5 years) and retrovirus positivity were risk factors for infection with the feline haemoplasmas in this study population. Further studies on feline haemoplasma infections in cats are needed to determine the significance of detecting small amounts of haemoplasma DNA, feline retrovirus infection and other associated risk factors on the clinical manifestation of disease.
doi:10.1186/1746-6148-8-103
PMCID: PMC3514208  PMID: 22748125
Feline haemoplasmas; qPCR; Reverse line blot; Feline retrovirus infection
12.  The prevalence of Bartonella, hemoplasma, and Rickettsia felis infections in domestic cats and in cat fleas in Ontario 
The prevalence of persistent bacteremic Bartonella spp. and hemoplasma infections was determined in healthy pet cats in Ontario. Blood samples from healthy cats sent to a diagnostic laboratory for routine health assessment over the course of 1 y were tested for Bartonella spp. using both polymerase chain reaction (PCR) and blood culture, and for the presence of hemoplasma by PCR. The overall prevalence of Bartonella spp. by PCR and by culture combined was 4.3% (28/646) [3.7% (24/646) Bartonella henselae, 0.6% (4/646) Bartonella clarridgeiae]. The novel B. henselae PCR developed for this study demonstrated nearly twice the sensitivity of bacterial isolation. The overall prevalence of hemoplasma was 4% (30/742) [3.3% (25/742) Candidatus Mycoplasma haemominutum, 0.7% (5/742) Mycoplasma haemofelis]. There was no significant difference between the prevalence of infection by season or by age (≤ 2 y, > 2 y). Candidatus Mycoplasma turicensis was identified, for the first time in Canada, in 1 cat. The prevalence of Bartonella (58%) and hemoplasma (47% M. haemofelis, 13% M. haemominutum) in blood from a small sampling (n = 45) of stray cats was considerably higher than that found in healthy pet cats.
The prevalence of Rickettsia felis in cat fleas was also assessed. A pool of fleas from each of 50 flea-infested cats was analyzed for the presence of R. felis by PCR. Rickettsia felis was confirmed, for the first time in Canada, in 9 of the 50 samples. Therefore, the prevalence of Bartonella and hemoplasma infection in healthy pet cats is relatively low. Further, the control of cat fleas is important because of the public health significance of Bartonella and R. felis infection.
PMCID: PMC2568045  PMID: 19086373
13.  Worldwide Occurrence of Feline Hemoplasma Infections in Wild Felid Species▿  
Journal of Clinical Microbiology  2007;45(4):1159-1166.
While hemoplasma infections in domestic cats are well studied, almost no information is available on their occurrence in wild felids. The aims of the present study were to investigate wild felid species as possible reservoirs of feline hemoplasmas and the molecular characterization of the hemoplasma isolates. Blood samples from the following 257 wild felids were analyzed: 35 Iberian lynxes from Spain, 36 Eurasian lynxes from Switzerland, 31 European wildcats from France, 45 lions from Tanzania, and 110 Brazilian wild felids, including 12 wild felid species kept in zoos and one free-ranging ocelot. Using real-time PCR, feline hemoplasmas were detected in samples of the following species: Iberian lynx, Eurasian lynx, European wildcat, lion, puma, oncilla, Geoffroy's cat, margay, and ocelot. “Candidatus Mycoplasma haemominutum” was the most common feline hemoplasma in Iberian lynxes, Eurasian lynxes, Serengeti lions, and Brazilian wild felids, whereas “Candidatus Mycoplasma turicensis” was the most prevalent in European wildcats; hemoplasma coinfections were frequently observed. Hemoplasma infection was associated with species and free-ranging status of the felids in all animals and with feline leukemia virus provirus-positive status in European wildcats. Phylogenetic analyses of the 16S rRNA and the partial RNase P gene revealed that most hemoplasma isolates exhibit high sequence identities to domestic cat-derived isolates, although some isolates form different subclusters within the phylogenetic tree. In conclusion, 9 out of 15 wild felid species from three different continents were found to be infected with feline hemoplasmas. The effect of feline hemoplasma infections on wild felid populations needs to be further investigated.
doi:10.1128/JCM.02005-06
PMCID: PMC1865832  PMID: 17301277
14.  Prevalence of Haemoplasma Infections in Stray Cats in Northern Italy 
ISRN Microbiology  2014;2014:298352.
This study investigated the prevalence of feline haemoplasma infections in a number of stray cat colonies in Milan, Northern Italy. Blood samples from 260 stray cats were evaluated, with conventional PCR, for the presence of DNA associated with Mycoplasma haemofelis (Mhf) and “Candidatus Mycoplasma haemominutum” (CMhm). Odd ratios (OR) were calculated to identify risk factors for haemoplasma infections. PCR was positive in 86 out of 260 subjects (33.1%), with a prevalence of 10.8% (28/260 cats) for Mhf and 22.3% (58/260 cats) for CMhm. No coinfections were registered. There were significant associations between infections and season of sampling, that is, a negative association between winter sampling and a haemoplasma positive status (OR = 0.29, P = 0.001), or CMhm positive status (OR = 0.29, P = 0.01). Haemoplasma infections are common in stray cats in Milan. Thus, domestic cats with outdoor access should be routinely monitored and treated for ectoparasites to minimize risks of disease acquisition. Moreover, as these infections are transmitted via blood, feline blood donors from this area should be screened by PCR and preferably be drawn from a population of indoor cats regularly treated for fleas.
doi:10.1155/2014/298352
PMCID: PMC3953429  PMID: 24707436
15.  Detection of feline haemoplasma species in experimental infections by in-situ hybridisation 
Microbial Pathogenesis  2011;50(2):94-99.
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”.
doi:10.1016/j.micpath.2010.09.003
PMCID: PMC3032890  PMID: 21129480
Haemoplasma; Mycoplasma haemofelis; “Candidatus Mycoplasma haemominutum”; “Candidatus Mycoplasma turicensis”; Fluorescence in-situ hybridisation
16.  Rapid detection of haemotropic mycoplasma infection of feline erythrocytes using a novel flow cytometric approach 
Parasites & Vectors  2013;6:158.
Background
The haemotropic mycoplasmas Mycoplasma haemofelis and Candidatus Mycoplasma haemominutum cause feline infectious anaemia with infection rates in feline populations reflecting widespread subclinical infection. Clinically significant infections are much rarer but can be life-threatening. Current diagnosis is dependent upon visualising organisms in stained blood smears, PCR or quantitative PCR (qPCR). These procedures are labour-intensive and time-consuming. Furthermore, PCR-based approaches offer limited insight into the disease burden of the infected animal.
Methods
We have developed a novel and rapid flow cytometric system that permits diagnosis of haemotropic mycoplasma infections and quantitation of the percentage of erythrocytes that are parasitized. The method exploits the fact that mature mammalian erythrocytes, the host cell for haemoplasmas, are enucleated and thus lack nucleic acid. DRAQ5 is a synthetic anthrocycline dye which rapidly crosses cell membranes and binds to nucleic acids. The presence of exogenous bacterial DNA in mammalian erythrocytes can, therefore, be detected by DRAQ5 uptake and flow cytometric detection of DRAQ5 fluorescence.
Results
Here, we show that this system can detect epi-erythrocytic infection of companion felines by haemotropic mycoplasma. Due to their differences in size, and hence the quantity of DNA, the two major feline hemoplasmas M. haemofelis and Candidatus M. haemominutum can be distinguished according to DRAQ5 fluorescence. We have also shown the usefulness of DRAQ5 uptake in monitoring a cat infected with M. haemofelis sequentially during treatment with doxycycline.
Conclusions
The technique described is the first report of a flow cytometric method for detecting haemotropic mycoplasmas in any species and could be applied to widespread screening of animal populations to assess infection by these epi-erythrocytic parasites.
doi:10.1186/1756-3305-6-158
PMCID: PMC3673821  PMID: 23725366
Hemoplasma; Hemotropic mycoplasma; Flow cytometry; Diagnosis
17.  Infection with Hemotropic Mycoplasma Species in Patients with or without Extensive Arthropod or Animal Contact 
Journal of Clinical Microbiology  2013;51(10):3237-3241.
PCR amplification targeting the 16S rRNA gene was used to test individuals with and without extensive arthropod and animal contact for the possibility of hemotropic mycoplasma infection. The prevalence of hemotropic mycoplasma infection (4.7%) was significantly greater in previously reported cohorts of veterinarians, veterinary technicians, spouses of veterinary professionals, and others with extensive arthropod exposure and/or frequent animal contact than in a previously reported cohort of patients examined by a rheumatologist because of chronic joint pain or evidence of small-vessel disease (0.7%). Based upon DNA sequence analysis, a Mycoplasma ovis-like species was the most prevalent organism detected; however, infection with “Candidatus Mycoplasma haematoparvum” and a potentially novel, but incompletely characterized, hemotropic Mycoplasma species was also documented. Historical exposure to animals and arthropod vectors that can harbor hemotropic Mycoplasma spp. should be considered during epidemiological investigations and in the evaluation of individual patients.
doi:10.1128/JCM.01125-13
PMCID: PMC3811635  PMID: 23863574
18.  First morphological characterization of ‘Candidatus Mycoplasma turicensis’ using electron microscopy 
Veterinary Microbiology  2011;149(3-4):367-373.
At least three haemotropic mycoplasmas have been recognized in cats: Mycoplasma haemofelis (Mhf), ‘Candidatus Mycoplasma haemominutum’ (CMhm) and ‘Candidatus M. turicensis’ (CMt). The latter was originally identified in a Swiss pet cat with haemolytic anaemia and shown to be prevalent in domestic cats and wild felids worldwide using molecular methods. So far, there has been no confirmatory morphological evidence of the existence of CMt presumably due to low blood loads during infection while CMhm has only been characterized by light microscopy with discrepant results. This study aimed to provide for the first time electron microscopic characteristics of CMt and CMhm and to compare them to Mhf. Blood samples from cats experimentally infected with CMt, CMhm and Mhf were used to determine copy numbers in blood by real-time PCR and for transmission and scanning electron microscopy. High resolution scanning electron microscopy revealed CMt and CMhm to be discoid-shaped organisms of 0.3 μm in diameter attached to red blood cells (RBCs). In transmission electron microscopy of CMt, an oval organism of about 0.25 μm with several intracellular electron dense structures was identified close to the surface of a RBC. CMhm and CMt exhibited similar morphology to Mhf but had a smaller diameter. This is the first study to provide morphological evidence of CMt thereby confirming its status as a distinct haemoplasma species, and to present electron microscopic features of CMhm.
doi:10.1016/j.vetmic.2010.11.020
PMCID: PMC3127424  PMID: 21183295
‘Candidatus Mycoplasma turicensis’; ‘Candidatus Mycoplasma haemominutum’; Haemoplasma; Haemotropic Mycoplasma; Electron microscopy; Real-time PCR
19.  Infectious disease prevalence in a feral cat population on Prince Edward Island, Canada 
The Canadian Veterinary Journal  2011;52(9):979-982.
Ninety-six feral cats from Prince Edward Island were used to determine the prevalence of selected infectious agents. The prevalence rates were 5.2% for feline immunodeficiency virus, 3.1% for feline leukemia virus, 3.1% for Mycoplasma haemofelis, 8.4% for Candidatus Mycoplasma haemominutum, 2.1% for Bartonella spp. and 29.8% for exposure to Toxoplasma gondii. Oocysts of T. gondii were detected in 1.3% of the fecal samples that were collected. Gender and retroviral status of the cats were significantly correlated with hemoplasma infections. Use of a flea comb showed that 9.6% of the cats had fleas; however, flea infestation was not associated with any of the infectious agents.
PMCID: PMC3157072  PMID: 22379197
20.  Development and Application of a Universal Hemoplasma Screening Assay Based on the SYBR Green PCR Principle▿  
Journal of Clinical Microbiology  2009;47(12):4049-4054.
Hemotropic mycoplasmas (hemoplasmas) are the causative agents of infectious anemia in several mammalian species. Their zoonotic potential has recently been substantiated by the identification of a feline hemoplasma isolate in an immunocompromised human patient. Although species-specific diagnostic molecular methods have been developed, their application as screening tools is limited due to the species diversity of hemoplasmas. The goals of this study were to develop a universal hemoplasma screening assay with broad specificity based on the SYBR green PCR principle, to compare the assay with hemoplasma-specific TaqMan PCR, and to analyze potential tick vectors and human blood samples to address the zoonotic potential. The newly developed PCR assay based on the 16S rRNA gene amplified feline, canine, bovine, porcine, camelid, and murine hemoplasmas, as well as Mycoplasma penetrans and Mycoplasma pneumoniae. The lower detection limit for feline and canine hemoplasmas was 1 to 10 copies/PCR. The assay exhibited 98.2% diagnostic sensitivity and 92.1% diagnostic specificity for feline hemoplasmas. All 1,950 Ixodes ticks were PCR negative, suggesting that Ixodes ticks are not relevant vectors for the above-mentioned hemoplasma species in Switzerland. None of the 414 blood samples derived from anemic or immunocompromised human patients revealed a clear positive result. The SYBR green PCR assay described here is a suitable tool to screen for known and so-far-undiscovered hemoplasma species. Positive results should be confirmed by specific TaqMan PCR or sequencing.
doi:10.1128/JCM.01478-09
PMCID: PMC2786680  PMID: 19828748
21.  Co-infection with Anaplasma platys, Bartonella henselae and Candidatus Mycoplasma haematoparvum in a veterinarian 
Parasites & Vectors  2013;6:103.
Background
During a two year period, a 27-year-old female veterinarian experienced migraine headaches, seizures, including status epilepticus, and other neurological and neurocognitive abnormalities. Prior to and during her illness, she had been actively involved in hospital-based work treating domestic animals, primarily cats and dogs, in Grenada and Ireland and anatomical research requiring the dissection of wild animals (including lions, giraffe, rabbits, mongoose, and other animals), mostly in South Africa. The woman reported contact with fleas, ticks, lice, biting flies, mosquitoes, spiders and mites and had also been scratched or bitten by dogs, cats, birds, horses, reptiles, rabbits and rodents. Prior diagnostic testing resulted in findings that were inconclusive or within normal reference ranges and no etiological diagnosis had been obtained to explain the patient’s symptoms.
Methods
PCR assays targeting Anaplasma spp. Bartonella spp. and hemotopic Mycoplasma spp. were used to test patient blood samples. PCR positive amplicons were sequenced directly and compared to GenBank sequences. In addition, Bartonella alpha Proteobacteria growth medium (BAPGM) enrichment blood culture was used to facilitate bacterial growth and Bartonella spp. serology was performed by indirect fluorescent antibody testing.
Results
Anaplasma platys, Bartonella henselae and Candidatus Mycoplasma haematoparvum DNA was amplified and sequenced from the woman’s blood, serum or blood culture samples. Her serum was variably seroreactive to several Bartonella sp. antigens. Despite symptomatic improvement, six months of doxycycline most likely failed to eliminate the B. henselae infection, whereas A. platys and Candidatus M. haematoparvum DNA was no longer amplified from post-treatment samples.
Conclusions
As is typical of many veterinary professionals, this individual had frequent exposure to arthropod vectors and near daily contact with persistently bacteremic reservoir hosts, including cats, the primary reservoir host for B. henselae, and dogs, the presumed primary reservoir host for A. platys and Candidatus Mycoplasma haematoparvum. Physicians caring for veterinarians should be aware of the occupational zoonotic risks associated with the daily activities of these animal health professionals.
doi:10.1186/1756-3305-6-103
PMCID: PMC3637287  PMID: 23587235
Bartonella; Mycoplasma; Anaplasma; Headache; Migraines; Seizures; Serology; PCR
22.  Chronic "Candidatus Mycoplasma turicensis" infection 
Veterinary Research  2011;42(1):59.
"Candidatus Mycoplasma turicensis" infects felids. The pathogenesis of "Candidatus M. turicensis" chronic infection is poorly understood. The goals of the present study were to (1) induce reactivation of the infection in chronic carrier cats by attempted immunosuppression, (2) identify potential tissue sequestration using real-time TaqMan® PCR and (3) monitor the humoral immune response by DnaK enzyme-linked immunosorbent assay (ELISA). Ten specified pathogen-free cats that had ostensibly recovered from experimental "Candidatus M. turicensis" infection were used: five cats (group 1) received high dose methylprednisolone (attempted immunosuppression), while five cats served as untreated controls (group 2). Besides weekly blood samples, tissue samples were collected from bone marrow, kidney, liver and salivary glands at selected time points. The cats in group 1 had significantly lower lymphocyte counts and higher blood glucose levels after methylprednisolone administration than the controls. After methylprednisolone administration one blood and three tissue samples from cats in group 1 tested PCR-positive; before the administration, only one sample was positive. All other samples tested PCR-negative. All cats stayed seropositive; the antibody levels of the cats in group 1 showed a significant transient decrease after methylprednisolone administration. This is the first study to report the presence of "Candidatus M. turicensis" in tissues of chronically infected cats and the persistence of anti-feline hemoplasma antibodies in the absence of detectable bacteremia. Methylprednisolone administration did not lead to a significant reactivation of the infection. Our results enhance the knowledge of "Candidatus M. turicensis" infection pathogenesis and are clinically relevant to the prognosis of hemoplasma-infected cats.
doi:10.1186/1297-9716-42-59
PMCID: PMC3090992  PMID: 21507220
23.  Description of outcomes of experimental infection with feline haemoplasmas: Copy numbers, haematology, Coombs’ testing and blood glucose concentrations 
Veterinary Microbiology  2009;139(3-4):323-332.
The aim of this study was to compare blood copy, haematological and glucose values between cats experimentally infected with either Mycoplasma haemofelis (Group HF: 10 cats), ‘Candidatus M. haemominutum’ (Group HM: 3 cats) or ‘Candidatus M. turicensis’ (Group TU: 3 cats). Blood samples were collected regularly up to 85 days post-infection (DPI) for haemoplasma real-time quantitative PCR, haematology, Coombs’ testing and blood glucose measurement. Statistical analysis was performed using a general linear model (ANOVA) appropriate for a repeated measures experiment with significance set as P < 0.05. Cats in Group TU had significantly lower blood copy numbers than cats in Group HF (P < 0.001) and HM (P < 0.001). All Group HF cats developed anaemia (often severe), macrocytosis and evidence of erythrocyte-bound antibodies whereas Groups HM and TU cats did not. Group HF had significantly lower PCVs, haemoglobin concentrations and red blood cell counts, and significantly higher mean cell volumes, than Groups HM and TU. In Group HF, erythrocyte-bound antibodies reactive at 4 °C (both IgM and IgG) appeared between 8 and 22 DPI and persisted for two to four weeks, whereas those reactive at 37 °C (primarily IgG) appeared between 22 and 29 DPI and persisted for one to five weeks. In most cats antibodies appeared after the fall in haemoglobin started. Although Group TU had significantly lower glucose concentrations than Groups HF (P = 0.006) and HM (P = 0.027), mean blood glucose concentrations remained within the reference range in all groups. This study demonstrates that M. haemofelis infection, in contrast to ‘Candidatus M. haemominutum’ and ‘Candidatus M. turicensis’ infection, can result in a severe macrocytic anaemia and the development of cold and warm reactive erythrocyte-bound antibodies.
doi:10.1016/j.vetmic.2009.06.028
PMCID: PMC2771275  PMID: 19615832
Haemoplasma; Quantitative real-time PCR; Coombs’ test; Autoagglutination; Glucose
24.  Molecular detection of hemoplasma infection among cats from São Luís island, Maranhão, Brazil 
Brazilian Journal of Microbiology  2012;43(2):569-575.
Hemoplasmas are bacteria that infect erythrocytes, attaching to the red blood cell. There is a need for more reports of hemoplasma infection prevalence and molecular characterization among cats in Brazil since there are only few published reports. The present work aimed to detect and molecularly characterize the presence of hemotrophic mycoplasmas in domestic cats with outdoor access from São Luís, Maranhão, Brazil. Twenty cats (10%) were positive for Candidatus M. haemominutum, five (2.5%) for M. haemofelis, and four (2.%) for M. turicensis based on 16S rRNA gene PCRs. Five cats (2.5%) were co-positive for Candidatus M. haemominutum and M. haemofelis. PCR diagnosis was confirmed by sequencing; and phylogenetic analysis was based on 16S rRNA and rnpb genes.
doi:10.1590/S1517-83822012000200018
PMCID: PMC3768807  PMID: 24031865
cats; Mycoplasma haemofelis; Candidatus Mycoplasma haemominutum; Candidatus Mycoplasma turicensis
25.  Absence of zoonotic Bartonella species in questing ticks: First detection of Bartonella clarridgeiae and Rickettsia felis in cat fleas in the Netherlands 
Parasites & Vectors  2011;4:61.
Background
Awareness for flea- and tick-borne infections has grown in recent years and the range of microorganisms associated with these ectoparasites is rising. Bartonella henselae, the causative agent of Cat Scratch Disease, and other Bartonella species have been reported in fleas and ticks. The role of Ixodes ricinus ticks in the natural cycle of Bartonella spp. and the transmission of these bacteria to humans is unclear. Rickettsia spp. have also been reported from as well ticks as also from fleas. However, to date no flea-borne Rickettsia spp. were reported from the Netherlands. Here, the presence of Bartonellaceae and Rickettsiae in ectoparasites was investigated using molecular detection and identification on part of the gltA- and 16S rRNA-genes.
Results
The zoonotic Bartonella clarridgeiae and Rickettsia felis were detected for the first time in Dutch cat fleas. B. henselae was found in cat fleas and B. schoenbuchensis in ticks and keds feeding on deer. Two Bartonella species, previously identified in rodents, were found in wild mice and their fleas. However, none of these microorganisms were found in 1719 questing Ixodes ricinus ticks. Notably, the gltA gene amplified from DNA lysates of approximately 10% of the questing nymph and adult ticks was similar to that of an uncultured Bartonella-related species found in other hard tick species. The gltA gene of this Bartonella-related species was also detected in questing larvae for which a 16S rRNA gene PCR also tested positive for "Candidatus Midichloria mitochondrii". The gltA-gene of the Bartonella-related species found in I. ricinus may therefore be from this endosymbiont.
Conclusions
We conclude that the risk of acquiring Cat Scratch Disease or a related bartonellosis from questing ticks in the Netherlands is negligible. On the other hand fleas and deer keds are probable vectors for associated Bartonella species between animals and might also transmit Bartonella spp. to humans.
doi:10.1186/1756-3305-4-61
PMCID: PMC3087693  PMID: 21501464

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