Design, Optimization, and Application of a Conventional PCR Assay With

Veterinary Clinical Pathology ISSN 0275-6382

ORIGINAL RESEARCH 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 Andrea P. Santos1, Joanne B. Messick2, Alexander W. Biondo3, Simone T. Oliveira1, Viviane Pedralli1, Camila S. Lasta1, Luciana A. Lacerda1, Vanessa S. Esteves1, Regina Hofmann-Lehmann4, Barbara Willi4, Felix´ H. D. Gonzalez´ 1

1Departamento de Patologia Clınica,´ Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil; 2Department of Veterinary Pathobiology, Purdue University, West Lafayette, IN, USA; 3Departamento de Medicina Veterinaria,´ Universidade Federal do Parana,´ Curitiba, Parana,´ Brazil; and 4Clinical Laboratory, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland

Key Words Background: ‘Candidatus Mycoplasma turicensis’ (CMtc) is a hemotrophic 28S rRNA gene, hemobartonellosis, bacterial species that can, alone or in combination, induce anemia in cats. hemoplasma, mycoplasma, PCR The diagnostic test of choice for hemoplasma infections is PCR. Conven- tional PCR assays have been developed for the detection of Mycoplasma Correspondence haemofelis (Mhf) and ‘Candidatus M. haemominutum’ (CMhm) but not for Andrea Santos, Department of Veterinary Pathobiology, Purdue University, 625 Harrison CMtc. Although real-time PCR assays have been reported for all of the fe- St., West Lafayette, IN 47907, USA line hemoplasmas, the expense of necessary instrumentation precludes its E-mail: [email protected] use in Brazil and many other countries. Objectives: The goals of this study were to develop and optimize a con- DOI:10.1111/j.1939-165X.2009.00158.x ventional PCR assay to diagnose CMtc using an internal control to detect false-negative results, and to evaluate the occurrence of CMtc infection in domestic cats from Brazil. Methods: Species-speciﬁc primers were designed and a PCR assay was developed for the detection of CMtc 16S rDNA in cat blood. Sensitivity was determined by serial 10-fold dilutions of plasmid and DNA extracted from blood from an experimentally infected cat. EDTA blood samples from 373 cats were collected. DNA was extracted using a silica-based protocol and tested using the PCR assay. Results: Primer concentration, annealing temperature, and MgCl2 concentration were optimized in the presence and absence of the internal control. Two samples negative for the internal control were excluded. Of the remaining 371 samples (117 healthy and 254 unhealthy cats), 17 (4.6%) were positive for CMtc. Conclusion: These results demonstrate the utility of an optimized PCR assay to detect CMtc in feline blood samples. We also report for the ﬁrst time the prevalence of CMtc infection in domestic cats in Brazil.

Introduction ‘Candidatus Mycoplasma haemominutum’ (CMhm) occasionally develop anemia, particularly when there Mycoplasma haemofelis (formerly known as Haemobar- is a concurrent disease.3 However, healthy cats exper- tonella felis – Ohio organism or large form) and ‘Candi- imentally infected with CMhm develop only minimal datus Mycoplasma haemominutum’ (formerly known clinical signs.4 as Haemobartonella felis – California organism or small A new species of hemotropic mycoplasma, ‘Candi- form) are hemotrophic bacterial species that parasitize datus Mycoplasma turicensis’ (CMtc), was described re- feline RBCs and can induce anemia.1,2 Acute infection cently as a potential cause of disease in cats. A with M. haemofelis (Mhf) is associated with severe naturally infected cat had severe hemolytic anemia hemolytic anemia. Cats that are naturally infected with and IV inoculation of the organism into an

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immunocompromised specific pathogen-free (SPF) cat M. coccoides (AY171918), M. haemomuris (U82963), also resulted in anemia.5 Phylogenetic analysis of the Bartonella henselae (Z11684), Bartonella clarridgeiae 16S rRNA gene of CMtc showed it was closely related (AB292603), Bartonella koehlerae (AF076237), and Bar- to Mycoplasma coccoides and Mycoplasma haemomuris, tonella quintana (AY484592) were downloaded from both of which are rodent hemotropic mycoplasmal the GenBank database22 and aligned using DNASTAR species.6 Since its first description, CMtc infection has Lasergene 7.1 (DNASTAR Inc., Madison, WI, USA) and been reported in domestic cats in the United States,7 CRUSTALW (http://www.ebi.ac.uk/Tools/clustalw/index. Switzerland,8,9 Japan,10 Germany,11 the United King- html).23 Potential target sites for forward primers dom, Australia, and South Africa6 and in wild felid were identified manually; those having a length of species from Spain, France, Switzerland, Tasmania, 18–24 bp, GC content of 35–60%, and melting temper- and Brazil.12 A paucity of information exists about the atures between 55 and 581C were investigated fur- epidemiology of CMtc. ther.24 Two forward primers were selected that had None of the hemoplasmas have been cultivated minimal stretches of polypurine or polypyrimidine or outside their natural hosts.13 Historically, the diagnosis stretches composed of the same base; these primers of infection has relied on microscopic identification were designated Mt1Fw and Mt2Fw (50-GTA TCC TCC of bacteria attached to RBCs. This method is neither ATC AGA CAG AA-30 and 50-AAC TGT CCA AAA GGC sensitive nor specific. It is well recognized that artifact AGT TA-30, respectively). Suitable reverse primers and such as stain precipitated on RBCs may be mistaken for PCR products were selected using the Primer3 program parasites and that parasites detach rapidly from the (http://primer3.sourceforge.net/).25 Based on the de- host cell in EDTA-anticoagulated blood.4,14 The sired product size (400–500 bp), melting temperatures, low parasite load in CMtc infection also is not easily minimal pair complementarity, and minimal pair 30 detected by light microscopy, further reducing the sen- complementarity (to avoid primer–dimer or hairpin sitivity of this method for diagnosis.9 Thus, PCR-based formation), 2 reverse primers were selected and were assays are the diagnostic method of choice for hemo- designated Mt1Rv and Mt2Rv (50-AGT ATT CGG CAC plasma infections. AAA CAA CT-30 and 50-CGC TCC ATA TTT AAT TCC The 16S rRNA gene is the basis for all hemoplasma AA-30, respectively). To verify the properties of each PCR assays to date, with different primer pairs and primer, PCR suitability tests were also done using the protocols reported for the 3 species infecting cats. SMS Sequence Manipulation Suite (http://bioinformatics. Conventional PCR assays have been developed for the org/sms2/index.html).26 detection of Mhf 14–16 and CMhm,4 but not for CMtc. The specificities of the prospective primers were Real-time PCR assays have also been developed to tested by basic local alignment search tool (blastn)27 detect and quantify feline hemoplasma species.9,17,18 against all existing DNA sequence information stored Using the feline 28S rRNA gene as an internal control, in GenBank. The selected oligonucleotide primers a duplex real-time assay was recently reported for each were synthesized commercially by IDT (Integrated of the hemoplasmas.19 The inclusion of an internal DNA Technologies, Coralville, IA, USA). control provides confirmation of the presence of amp- lifiable DNA and/or the absence of inhibitors in the Controls sample.20,21 The overall expense of purchasing real- time PCR instrumentation precludes its use in Brazil Previously described plasmid containing almost the and many other countries. Therefore, the aim of this entire CMtc 16S rRNA gene (plasmid control) and research was to develop and apply a conventional PCR DNA extracted from 200 mL of EDTA blood from an assay for the diagnosis of CMtc infection that can be SPF cat experimentally infected with CMtc (genomic used alone or as a duplex PCR with the feline-specific DNA control) were used as DNA templates.5 Both con- 28S rRNA gene as an endogenous control. Using trols were tested previously using a quantitative real- the optimized assay protocol, the prevalence of CMtc time PCR assay and were shown to have 105 and infection in domestic cats in Brazil was determined. 1103.18 (plasmid and genomic DNA controls, respectively) copies of the organism per 5 mL of reaction vol- Material and Methods ume. Concentration and purity of the CMtc controls were also evaluated by UV spectrophotometry using Nanodrop ND-1000 (Nanodrop Technologies, Wil- Primer design and selection mington, DE, USA). The concentration of nucleic Nucleotide sequences for the 16S rRNA gene of CMtc acids was calculated from the optical density (OD) at (DQ157153), Mhf (U95297), CMhm (AM745338), 260 nm, whereas the ratio between the absorbance at

444 Vet Clin Pathol 38/4 (2009) 443–452 c 2009 American Society for Veterinary Clinical Pathology Santos et al PCR assay for ‘‘Candidatus Mycoplasma turicensis’’

260 and 280 nm (OD260/OD280) and a continuous scan Validation of primer specificity of UV absorbance from 220 to 320 nm provided an es- Primer specificity was defined as the ability of a primer timate of sample purity. Negative controls included to amplify specifically CMtc. Specificities of the primers DNA extracted from a noninfected cat previously were tested against Mhf, CMhm, and B. henselae (types tested against the 3 feline hemoplasmas and autoc- I, II, and Houston I strains). The B. henselae positive laved ultrafiltered water. controls were kindly provided by Dr. Lynn Guptill at Purdue University. To confirm the identity of the PCR Conventional PCR for CMtc products, the purified PCR amplicons (PureLink PCR Each PCR mixture consisted of 1 Green GoTaq Flexi Purification Kit, Invitrogen) were cloned into the Buffer (pH 8.5), 1.5 mM MgCl2, deoxynucleoside pGEM-T EasyVector (Promega). To minimize the pos- triphosphates (dNTPs) at a concentration of 200 mM, sibility of sequencing errors attributable to misincor- group-specific primers at a concentration of 0.4 mM, porations by the GoTaq Flexi DNA Polymerase, each 1.25 U of GoTaq Flexi DNA Polymerase (Promega, strand of the insert was submitted for sequencing from Madison, WI, USA), template DNA (5 mL), and auto- 3 different clones. Plasmid DNA was purified with a claved ultrafiltered water to a total volume of 25 mL per QIAprep Spin Miniprep kit (Qiagen, Valencia, CA, reaction. The PCR, carried out in an Eppendorf Mas- USA). Sequence authenticity of the cloned amplicons tercycler gradient thermocycler (Eppendorf Scientific was verified by dideoxy DNA sequencing (Purdue Inc., Westbury, NY, USA), consisted of 1 cycle of 951C Genomics Core Facility at Purdue University, West for 2 minutes; 35 cycles of 941C for 1 minute; 551C for Lafayette, IN, USA) followed by alignment with their 45 seconds; 721C for 45 seconds; and 1 cycle of 721C for corresponding sequences in GenBank. 5 minutes. The amplification products were electro- phoresed in a 1.5% agarose gel for 1 hour at 80 V, fol- Internal control using 28S rRNA gene lowed by ethidium bromide (1 mg/mL) staining and vi- Except for the primers, a singleplex PCR using the sualization under UV light. Gels were photographed same conditions as for the CMtc PCR assay was first using Epi Chemi II Darkroom (UVP Inc., Upland, CA, applied to amplify the feline 28S rRNA gene. The prim- USA). A 100-bp DNA ladder (Invitrogen, Carlsbad, CA, ers used (feline 28S rDNA Fw 50-AGC AGG AGG TGT USA) was used to compare product sizes. TGG AAG AG-30 and feline 28S rDNA Rv 50-AGG GAG AGC CTA AAT CAA AGG-30) were previously Determination of the limit of detection 31 described by Helps and collaborators. Although the The lower limit of detection was defined as the smallest feline 28S rDNA has been used in real-time PCR as- number of organisms in a sample that could be de- says,19,32 we evaluated 371 feline samples from Brazil tected by the new PCR assay.28 This was determined by for 28S rDNA (DNA extracted from blood) and com- 10-fold serial dilutions of the plasmid and genomic pared the results with the housekeeping gene target DNA controls and considering 1–2 copies of the 16S glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rRNA gene per organism.29 to validate its use in the conventional PCR assay.33 The GAPDH protocol consisted of 1 Green GoTaq Flexi Optimization Buffer (pH 8.5), 1.5 mM MgCl2, dNTPs at a concentration of 200 mM, group-specific primers at a concentra- In addition to the selection of primers, 3 experiments tion of 0.2 mM, 1.25 U of GoTaq Flexi DNA polymerase, were performed to optimize the PCR protocol for: (1) template DNA (5 mL), and autoclaved ultrafiltered optimal primer concentration, using a range from 0.08 water to a total volume of 25 mL per reaction. The 29 to 0.6 mM for each primer tested ;(2)optimalanneal- thermocycler conditions consisted of 1 cycle of 941C ing temperature (Ta), by setting the gradient thermo- for 5 minutes; 30 cycles of 941C for 45 seconds; 551C for cycler at 21C increments from 45.3 to 65.61Cand 45 seconds; 721C for 45 seconds; and 1 cycle of 721C for estimating the primer annealing temperature using the 5 minutes. OPT oligo-PCR Ta Calculator program (http://webu.co.uk/ oligo/index.php); and (3) optimal MgCl concentration, 2 Duplex PCR for CMtc and feline 28S rRNA gene using a range from 0.5 to 4.0 mM and measuring the yield of PCR products and fidelity of the PCR assay.30,31 Using the same conditions as the singleplex CMtc pro- For all 3 experiments, the most diluted, positive samples tocol, a duplex PCR was evaluated by adding the feline of plasmid and genomic DNA templates were used in 28S rDNA primers to the protocol. To avoid competi- addition to nondiluted samples. tion for reagents and loss of target sensitivity, a primer

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balance experiment was performed starting with equi- evaluated by dividing the cats into 3 groups: o5, 6–10, molar amounts of CMtc and 28S rDNA primers (0.4 mM and 411 years old. Associations were considered sta- each) and adjusting the concentration of the internal tistically significant when P o.05. primer in the duplex assay.24 Also, 10-fold serial dilutions using control materials and controls made using Results half of the CMtc-positive genomic control and half of DNA extracted from a noninfected cat were performed Primer choice and specificities to check the sensitivity of the assay. PCR products having the predicted sizes were obtained PCR assay in naturally infected cats for the CMtc 16S rRNA gene from plasmid and genomic DNA controls for all combinations of primers The optimized PCR assay was evaluated by testing 373 (Mt1Fw–Mt1Rv, Mt1Fw–Mt2Rv, Mt2Fw–Mt1Rv, and blood samples from cats in Brazil, including nonhospi- Mt2Fw–Mt2Rv). However, the 2 sets containing the talized pet cats from Porto Alegre being screened for Mt2Fw primer gave rise to primer–dimer and/or hair- blood donation with the owner’s consent (n = 90), cats pin formation as seen faintly on the gel and as pre- presented to the Veterinary Hospital at Universidade dicted by the SMS program.26 A strong band of Federal do Rio Grande do Sul, Porto Alegre, Rio amplified product was observed for plasmid control us- Grande do Sul, Brazil, between January 2006 and ing the primer set, Mt2Fw–Mt2Rv; however, the prod- May 2007 (n = 263), and feral cats housed in a local uct yield was low or absent for the genomic DNA shelter (n = 20). The cats were divided into 2 groups control. Thus, the Mt2Fw primer was excluded from based on the results of a physical examination, CBC, further evaluation. Comparing the remaining sets of and chemistry profile. Group 1 consisted of 118 clini- primers (Mt1Fw–Mt1Rv and Mt1Fw–Mt2Rv), nonspe- cally healthy cats (68 pet, 40 hospitalized, and 10 fe- cific bands were observed with the set Mt1Fw–Mt1Rv ral); results of the physical examination and laboratory when tested against the genomic DNA extracted from tests were unremarkable in these cats. Group 2 con- some Brazilian cats, a finding not observed in the ge- sisted of 255 cats that were considered unhealthy (22 nomic DNA control (data not shown). The observed pet, 223 hospitalized; and 10 feral) as determined by absence of nonspecific amplification and lack of dimers any abnormal findings on physical examination or lab- contributed to selection of primer set Mt1Fw–Mt2Rv oratory tests. for development of our conventional PCR assay. Fur- DNA was extracted from EDTA whole blood samples ther, when used together, Mt1Fw and Mt2Rv, based on using a silica-based protocol34 and stored at 201Cfor database searches, would specifically amplify the 16S up to 2 months until PCR testing was performed. Ampli- rDNA of CMtc and when tested with Mhf, CMhm, and fication products of the positive samples for CMtc were Bartonella sp. positive controls, no amplified products purified from the agarose gel using the Zymoclean Gel were seen (data not shown). Thus, the primer set, DNA Recovery Kit (Zymo Research, Orange, CA, USA), Mt1Fw–Mt2Rv, was shown to be specific for amplifi- cloned into the pGEM-T EasyVector and sequenced. To cation of the 16S rDNA of CMtc from genomic DNA evaluate coinfection with other hemoplasmas, previ- and plasmid controls. Figure 1 shows the multiple ously described PCR assays for the detection of Mhf 15 alignments between closely related hemoplasma spe- and CMhm4,14 were also performed. cies and the locations of primer annealing in the CMtc sequence. Statistical analysis Prevalence was calculated by dividing the number of Limit of detection occurrences of infection during the specified time pe- riod by the size of the population in which the infec- Amplification of CMtc plasmid and genomic DNA con- tion occurred. A descriptive analysis of the variable trols was efficient out to 106 (0.2 copies/mL) and 104 (positive/negative for CMtc infection) was performed (0.11 copies/mL) dilutions of the starting template, re- for each group. spectively. The initial concentration of the CMtc plas- Chi-squared test or Fisher’s exact test (when ob- mid control DNA evaluated by UV spectrophotometry servations were 5) was used for univariate analysis was 19.51 ng/mL, with an OD260/OD280 ratio of 1.76 of selected variables. Data were compiled and analyzed and scan results (UV absorbance from 220 to 320 nm) in SAS version 9.1 (SAS Institute Inc., Cary, NC, USA). consistent with pure DNA. When evaluating the ge- The variables included: source (n = 371), gender nomic DNA control, the concentration was 17.04 ng/

(n = 317), breed, and variable age (n = 107). Age was mL; however, the low OD260/OD280 ratio of 1.25 and

446 Vet Clin Pathol 38/4 (2009) 443–452 c 2009 American Society for Veterinary Clinical Pathology Santos et al PCR assay for ‘‘Candidatus Mycoplasma turicensis’’

Figure 1. Multiple sequence alignment of hemotrophic mycoplasma species using the Clustal W program. GenBank accession numbers and species are shown. Primer locations for the ‘Candidatus Mycoplasma turicensis’ PCR assay developed in this study are underlined.

UV scan indicated protein, solvent, or buffer contami- established above) and dNTPs were used at standard nation of the sample (data not shown). concentrations of 200 mM each.24 In the presence of

200 mM of each dNTP, a series of MgCl2 titrations in 0.5 mM increments were done over the 0.5–4.0 mM Optimization concentration range to determine the MgCl2 concen- The amount of each primer was tested using the con- tration that produced the highest yield of a specific centration range of 0.08–0.6 mM. Primer concentra- PCR product. Using 0.5 mM MgCl2, diminished band tions from 0.25 to 0.6 mM were shown to amplify both intensity was obtained compared with band intensities plasmid and genomic DNA control templates (data not seen with 1.0–3.5 mM and nonspecific product ampli- shown). However, when a smaller amount of primer fication appeared at a MgCl2 concentration of 4.0 mM. was used ( o 0.2 mM) weak bands were observed. No Thus, we used a standard MgCl2 concentration of evidence of primer accumulation was observed even at 1.5 mM for the CMtc PCR protocol. the highest primer concentration of 0.6 mM. Based on these results, a primer concentration of 0.4 mM was Duplex PCR used in subsequent experiments. Except for inclusion of the feline 28S rDNA primers, The annealing gradient experiment was per- the same conditions as described previously for the formed at temperatures between 45.31C and 65.61C. singleplex CMtc protocol were used. The concentration At annealing temperatures between 45.31C and 61.01C, of primers used for amplifying the 16S rDNA of CMtc PCR product was amplified from the genomic DNA was 0.4 mM and the concentration of feline 28S rDNA control diluted to 102 and plasmid control diluted to primers was incrementally decreased to 0.06 mM. At a 106 (data not shown). Relative intensities of specific primer concentration of 0.4 mM for CMtc and 0.08 mM PCR products amplified from the diluted genomic DNA for feline 28S rDNA, distinct bands with the size control began to decline when the annealing temper- predicted for both CMtc (488 bp) and feline 28S rDNA ature was 4 58.51C (data not shown). At temperatures (100 bp) were obtained. In addition, neither non- higher than 63.11C no PCR products were found in the specific bands nor primer–dimer formation were 1.5% ethidium bromide agarose gel. observed. As with singleplex PCR, genomic DNA of PCR products in a single band (specific amplifica- the positive control could be detected out to a 104 di- tion) were found for T at 53.0–61.01C. Amplification a lution of starting template in the duplex assay. Ampl- of products was found at or below annealing tempera- icons of both the 16S rRNA and feline 28S rRNA tures of 63.11C; however, a pattern of nonspecific prod- genes were still detected at this dilution (Figure 2). uct amplification appeared at or below an annealing The same results between singleplex and duplex were temperature of 50.41C. While a conclusive reason for observed when the same positive DNA control was this nonspecific amplification product was not deter- serially diluted with blood from a noninfected cat mined, the results were reproducible. Thus, using (mixed control). Mt1Fw and Mt2Rv, the optimum Ta for the PCR was 53–55.81C. PCR assay in naturally infected cats In the MgCl2 experiment, the concentrations of all reagents in the PCR protocol were kept constant Group 2 cats were significantly older and more likely

(primer concentration of 0.4 mM and Ta of 551Cas to be male than Group 1 cats (Table 1). Four of 117

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Table 1. Source, gender, breed, age, and outdoor access of healthy and unhealthy cats from Porto Alegre, Rio Grande do Sul, Brazil. No. (%) of Cats

Variable Healthy Unhealthy P ValueÃ Source (n = 371) Pet population 68 (58.1) 22 (8.7) o .0001 Hospitalized population 39 (33.4) 222 (87.4) Feral, at shelter 10 (8.5) 10 (3.9) Gender (n = 317) Male 51 (45.5) 123 (60.0) .0134 Female 61 (54.5) 82 (40.0) Breed (n = 203) Figure 2. Comparison between the singleplex PCR assay for CMtc (lanes Pedigree 15 (13.6) 16 (17.2) .4813 2–6) and the duplex PCR assay for CMtc and feline 28S rDNA (lanes 7–11) Nonpedigree 95 (86.4) 77 (82.8) at different dilutions. Lane 1: 100 bp DNA ladder; lanes 2 and 7: genomic Age (n = 107) DNA control sample extracted from blood; lanes 3–5 and 8–10: genomic 5 years 35 (32.7) 38 (35.5) .0033 DNA control in dilutions of 102,103, and 104; lanes 6 and 11: genomic o 6–10 years 6 (5.6) 16 (14.9) DNA from a noninfected cat; lane 12, negative control (ultrapure water). 4 11 years 0 (0) 12 (11.2) CMtc, ‘Candidatus Mycoplasma turicensis.’ ÃChi-squared test was used to assess significant differences between healthy and unhealthy cats based on source, gender, and breed. Fisher’s exact test was used for the variable age. (3.4%) CMtc-positive cats were healthy and 13 of 254 (5.11%) were unhealthy. However, the unhealthy cats were not statistically more likely to have CMtc infection (P 4.05). number EU580599). Sequencing for 1 of the other The PCR assay developed in this study was per- 2 clones failed. The remaining sequence matched formed on extracted DNA from 373 EDTA blood sam- with CMtc; however, it was not 100% homologous ples from cats (Table 2). Two samples were negative for with either Porto Alegre 1 or 2. the feline 28S rRNA gene target and were excluded from the study. Of the remaining 371 samples, 79 (21.3%) were PCR-positive for at least 1 species of Discussion hemoplasma. Seventeen of 371 (4.6%) samples were positive for CMtc; 5 of these 17 were coinfected with In the present study, an internally controlled conven- CMhm and 2 were coinfected with CMhm and Mhf. tional PCR assay that targeted the 16S rRNA gene for The prevalence of Mhf and CMhm was 2.16% and diagnosis of CMtc infection in cats was designed, opti- 13.48%, respectively. Four of 371 (1.1%) cats were mized, and evaluated in clinical samples. Although coinfected with Mhf and CMhm. real-time PCR-based fluorescence assays have advan- Fragments of 488 bp purified from the agarose gel tages over conventional PCR, the former requires spe- from 15 of the positive samples were cloned, se- cialized instrumentation that is not readily available in quenced, analyzed, and compared with known se- Brazil and other countries. Identification of the specific quences in the GenBank database. An insufficient hemoplasma species is important to our understanding amount of DNA was extracted from 2 samples, pre- of the epidemiology and clinical presentation of infec- cluding their sequencing. Eleven of the sequences tion in cats. While Mhf has been reported to cause se- were identical to each other and also showed 100% vere hemolytic anemia in immunocompetent cats, homology with 9 of 22 CMtc sequences in the Gen- CMhm infection is associated with minimal clinical Bank database. Our sequence was designated ‘‘isolate signs.4,35 CMtc, a newly recognized hemoplasma, may Porto Alegre 1’’ and submitted to the GenBank da- have a lower hemolytic capacity than Mhf, but anemia tabase (accession number EU580598). Two of the may be worse in cats that are immunocompromised or sequences had a total of 10 nucleotides, which were have a coinfection. Other than a single case report, the not in common with the 10 previously mentioned presence and clinical importance of CMtc infection of sequences but were 100% homologous with 3 se- cats in Brazil has not been investigated to date. This is quences in the database (DQ464425, DQ464424, the first study using a conventional PCR assay for the DQ464423). These sequences were designated ‘‘isolate diagnosis of CMtc and to document the occurrence of Porto Alegre 2’’ and submitted to GenBank (accession infection in domestic cats in Brazil.

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Table 2. Prevalence estimates for hemoplasma infection based on pos- DNA template the appearance of these bands and per- itivity using a PCR assay in healthy and unhealthy cats from Porto Alegre, haps even more nonspecific secondary products would Rio Grande do Sul, Brazil. be anticipated at low annealing temperatures. This No. (%) of PCR-Positive Cats experiment was not performed because of the limited amount of template DNA (genomic DNA control). Healthy Unhealthy Feline Hemoplasma (n = 117) (n = 254) P ValueÃ The gradient experiment also showed that the Mt1Fw–Mt2Rv primers gave specific product amplifi- Hemoplasmasw 23 (19.7) 56 (22.0) .6015 1 Mhf 2 (1.7) 6 (2.4) 1.000 cation (488 bp) for annealing temperatures of 53–63 C. CMhm 15 (12.8) 35 (13.7) .9203 However, band intensities began to decrease at CMtc 4 (3.4) 6 (2.7) .5146 annealing temperatures 4 58.51C. An optimum an- OPT Mhf/CMhz 1 (0.9) 3 (1.2) .6240 nealing temperature (Ta ) was calculated based on CMhm/CMtc‰ 1 (0.9) 4 (1.6) 1.000 thermodynamic parameters and base composition36 Mhf/CMhm/CMtcz 0 (0) 2 (0.8) 1.000 OPT using the oligo-PCR Ta Calculator program. The Ã OPT Fisher’s exact test was used to assess significance differences in hemo- T a for the CMtc protocol was 55.221C and for the fe- plasma infection between healthy and unhealthy cats. line 28S rDNA protocol was 54.831C. On the basis of wPositive for at least 1 hemoplasma DNA; double or triple infected the combined experimental and calculated results, an counted only once. annealing temperature of 551C was selected for our zCoinfected with M. haemofelis and ‘Candidatus M. haemominutum.’ PCR assay. ‰Coinfected with ‘Candidatus M. haemominutum’ and ‘Candidatus M. DNA polymerase activity requires an optimal turicensis.’ 21 21 zCoinfected with M. haemofelis,‘Candidatus M. haemominutum,’ and concentration of free Mg . Because Mg is bound ‘Candidatus M. turicensis.’ by dNTPs and nucleotide template, the CMtc PCR with low and high amounts of template was optimized by

gradually increasing MgCl2 concentration from 0.5 to Primer design is critical to the yield and fidelity of 4 mM while keeping the concentration of dNTPs at PCR. Although the nucleotide sequence in some re- 200 mM each.29 It was not surprising that nonspecific gions of 16S rDNA is highly conserved, other regions amplification products appeared at high concentra- are sufficiently divergent and can be used as targets for tions of MgCl2, whereas lower concentrations im- designing primers that will provide species-specific proved the specificity of the PCR.30 Magnesium ion amplification. In this study, 3 freely available web- concentrations of 1.0–3.5 mM produced the highest based programs, Primer 3, Sequence Manipulation yield of specific PCR product. Thus, based on these re- Suite, and blastn, were used to aid in the selection of sults and convention, we decided to use a concentra- primers for specific amplification of CMtc. Although tion of 1.5 mM for the reaction. the 2 forward primers, Mt1Fw and Mt2Fw, were man- Both the singleplex and duplex PCR assays were ually identified in aligned sequences, suitable reverse used to test for CMtc DNA in blood samples from cats in primers for the PCR, Mt1Rv and Mt2Rv, were selected southern Brazil. To evaluate the duplex assay, all sam- using the Primer 3 program. Properties of these prim- ples that were positive in the singleplex PCR and 52 ers, including melting temperature, percent GC con- negative samples were tested. The remainder of the tent, and PCR suitability, were verified using Sequence samples negative by singleplex PCR were not tested Manipulation Suite: PCR Primer Stats. A trial version because of limited sample volume. A limited amount of a commercially available program was used for mul- of feline 28S rDNA primers was used to minimize com- tiple sequence alignments; however, free software such petition between amplification of DNA targets. A as ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/ primer concentration of 0.06 mM was enough to am- index.html) can be downloaded on a local computer plify the 28S rRNA gene in the 371 positive samples for to perform this function. Finally, blastn was run to as- the GAPDH housekeeping gene. Nonetheless, the certain that the primer sequences were unique within same results were observed when comparing the sin- the template sample to be amplified. gleplex and duplex assays for all of these samples and In the temperature gradient experiment, nonspe- when 10-fold dilutions of positive samples were tested. cific amplification was observed at low genomic DNA In our study population, the overall prevalence of template concentrations when annealing temperature CMtc infection as detected by conventional PCR was was below 531C. It is likely these nonspecific products 4.6% (17 cats). This result was somewhat higher than were due to amplification of cat genomic DNA because that reported by authors sampling similar mixed pop- nonspecific amplification was not observed using a ulations of healthy and sick cats in Switzerland (1%),8 plasmid control. In the presence of higher amounts of Germany (2.25%),11 and the United Kingdom

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(2.3%).6 The high seasonal prevalence of potential Acknowledgments vectors in Porto Alegre, a subtropical location, might We would like to thank the Coordenaçaõ de Aper- in part explain these results. However, the occurrence feiçoamento de Pessoal de N´ıvel Superior (CAPES) for the of CMtc infection in unhealthy cats was lower in this financial support for this study and for Dr. Andrea Santos’s study (5.11%) than those reported by others in personal research fellowship; Ching-Yun Chang at Purdue 7 6 the United States (6.5%) and South Africa (26%) University for statistical analysis support; Dr. Ana Paula using populations of cats suspected to be infected or Ravazollo and Dr. Claudio´ W. Canal at Universidade Federal sick and presented to a veterinary hospital. The major- do Rio Grande do Sul; and the Department of Comparative ity (139/147) of cats in a study in Australia also were Pathobiology, School of Veterinary Medicine at Purdue sick and had an occurrence of CMtc infection of 10%.6 University, USA, for mentoring and laboratory support. The lack of significant difference in hemoplasma prevalence between healthy and unhealthy cats in our study was in line with a similar study of cats in Swit- References zerland8 and could imply that the pathogenic potential of the parasites is not related to the presence of con- 1. Neimark H, Johansson KE, Rikihisa Y, Tully G. Proposal current diseases. We can conclude that CMtc infection to transfer some members of the genera Haemobartonella and Eperythrozoon to the genus is present in the Brazilian domestic cat population; Mycoplasma with descriptions of ‘Candidatus however, unhealthy cats are not more likely to be Mycoplasma haemofelis,’ ‘Candidatus Mycoplasma infected. haemomuris,’ ‘Candidatus Mycoplasma haemosuis’ and Two different CMtc strains, Porto Alegre 1 ‘Candidatus Mycoplasma wenyonii.’ Int J Syst Evol (EU580598) and Porto Alegre 2 (EU580599), were Microbiol. 2001;51:891–899. identified in this study and submitted to the GenBank 2. Messick JB. Hemotrophic mycoplasmas database. Eleven of the 448 bp fragments from the 16S (hemoplasmas): a review and new insights into rDNA sequenced were identical to each other and ho- pathogenic potential. Vet Clin Pathol. 2004;33:2–13. mologous to 9 of 22 sequences in Genbank, including that of an experimentally infected cat from Switzer- 3. De Lorimier LP, Messick JB. Anemia associated with land, which we used as our positive control. Isolate ‘Candidatus Mycoplasma haemominutum’ in a feline Porto Alegre 1 was more common in our study and leukemia virus-positive cat with lymphoma. JAm Anim. 2004;40:423–427. corresponded to the major cluster previously described by Willi et al.6 Two sequences (Porto Alegre 2) 4. Foley JE, Harrus S, Poland A, Chomel B, Pedersen NC. were 100% identical but had 10 nucleotide differences Molecular, clinical and pathologic comparison of two when compared with Porto Alegre 1. It has distinct strains of Haemobartonella felis in domestic cats. recently been shown by 16S rDNA sequence analysis Am J Vet Res. 1998;59:1581–1588. that some Australian (DQ464425 and DQ464423) and 5. Willi B, Boretti FS, Cattori V, et al. Identification, South African (DQ464424) CMtc isolates at molecular characterization, and experimental GenBank appear to branch away from the remaining transmission of a new hemoplasma isolate from a cat isolates.6 The isolate Porto Alegre 2 had 100% homo- with hemolytic anemia in Switzerland. J Clin Microbiol. logy with these divergent sequences, suggesting that at 2005;43:2581–2585. least 2 strains of CMtc exist. 6. Willi B, Tasker S, Boretti FS, et al. Phylogenetic analysis In summary, the conventional PCR assay de- of ‘Candidatus Mycoplasma turicensis’ isolates from pet scribed herein is a reliable tool for the diagnosis of cats in the United Kingdom, Australia, and South CMtc infection in cats. As a singleplex assay or when Africa, with analysis of risk factors for infection. J Clin duplexed in the same tube with an internal control, Microbiol. 2006;44:4430–4435. the assay provides a sensitive, specific, and rapid 7. Sykes JE, Terry JC, Lindsay LL, Owens SD. Prevalences method that can be easily implemented in diagnostic of various hemoplasma species among cats in the and research laboratories. This is especially important United States with possible hemoplasmosis. JAmVet in countries where the expense of real-time PCR in- Med Assoc. 2008;1232:372–379. strumentation precludes its use. This is the first report 8. Willi B, Boretti FS, Baumgartner C, et al. Prevalence, of prevalence of hemoplasma infections in domestic risk factor analysis, and follow-up of infections caused cats in Brazil; infection with CMtc was common. Our by three feline hemoplasma species in cats in laboratory is currently using the assay to investigate Switzerland. J Clin Microbiol. 2006;44:961–969. the clinical importance and risk factors of CMtc and 9. Willi B, Boretti FS, Baumgartner C, et al. Feline other hemoplasma infections in cats in Brazil. hemoplasmas in Switzerland: identification of a novel

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