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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 de- veloped 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 con- centration were optimized in the presence and absence of the internal con- trol. 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 as- say 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 Vet Clin Pathol 38/4 (2009) 443–452 c 2009 American Society for Veterinary Clinical Pathology 443 PCR assay for ‘‘Candidatus Mycoplasma turicensis’’ Santos et al 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, respec- tively) 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.
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