Detection of Mixed Infections with "Candidatus Mycoplasma Haemominutum" and Mycoplasma Haemofelis Using Real-Time TaqMan Polymerase Chain Reaction

Jane E Sykes, Nicole L. Drazenovich, Andrew E. Kyles, Louise M. Ball, Christian M. Leutenegger

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

The objective of this study was to evaluate the use of real-time TaqMan PCR assays for detection of coinfections with "Candidatus Mycoplasma haemominutum" (Mhm), and Mycoplasma haemofelis (Mhf), in vitro and over time in experimentally infected cats. First, the ability of each real-time PCR assay to detect and quantify mixed infections was determined in vitro by testing mixtures of plasmids containing Mhm and Mhf 16S rDNA with each assay. Subsequently, 4 specific pathogen-free (SPF) cats, 2 of which were splenectomized, were inoculated with blood from a cat infected with both Mhm and Mhf. Sixteen blood samples were then collected from each cat over a 55-day period. Each of the 64 postinoculation samples was tested using both conventional polymerase chain reaction (cPCR) and real-time PCR for the 16S rRNA gene of each organism. When applied to mixtures of plasmid DNA from each species, the results of quantitation with each of the real-time PCR assays approximately reflected the number of plasmid copies present. Forty-nine of 64 post-inoculation samples (77%) were positive using both cPCR and real-time PCR, 4 (6%) were positive using cPCR only, and 3 (5%) were positive using real-time PCR only. Both organisms were detected in 23 samples using real-time PCR. Mixed infections were not detected using cPCR. The size of the corresponding cPCR products suggested infection with Mhm in 4 and Mhf in 18 of these samples. The use of multiple separate real-time PCR assays rather than cPCR alone should thus be considered for epidemiologic studies of hemoplasmosis in cats.

Original languageEnglish (US)
Pages (from-to)250-255
Number of pages6
JournalJournal of Veterinary Diagnostic Investigation
Volume19
Issue number3
DOIs
StatePublished - 2007

Fingerprint

Mycoplasma haemominutum
Mycoplasma haemofelis
Mycoplasma
Coinfection
mixed infection
Real-Time Polymerase Chain Reaction
quantitative polymerase chain reaction
polymerase chain reaction
Cats
Polymerase Chain Reaction
cats
assays
plasmids
Plasmids
sampling
Mycoplasma Infections
Specific Pathogen-Free Organisms
blood
organisms
Ribosomal DNA

Keywords

  • Anemia
  • Haemobartonella
  • hemoplasma
  • quantitative PCR
  • splenectomy

ASJC Scopus subject areas

  • veterinary(all)
  • Microbiology

Cite this

Detection of Mixed Infections with "Candidatus Mycoplasma Haemominutum" and Mycoplasma Haemofelis Using Real-Time TaqMan Polymerase Chain Reaction. / Sykes, Jane E; Drazenovich, Nicole L.; Kyles, Andrew E.; Ball, Louise M.; Leutenegger, Christian M.

In: Journal of Veterinary Diagnostic Investigation, Vol. 19, No. 3, 2007, p. 250-255.

Research output: Contribution to journalArticle

@article{46dcac3fc6ef4467b01658885cca694d,
title = "Detection of Mixed Infections with {"}Candidatus Mycoplasma Haemominutum{"} and Mycoplasma Haemofelis Using Real-Time TaqMan Polymerase Chain Reaction",
abstract = "The objective of this study was to evaluate the use of real-time TaqMan PCR assays for detection of coinfections with {"}Candidatus Mycoplasma haemominutum{"} (Mhm), and Mycoplasma haemofelis (Mhf), in vitro and over time in experimentally infected cats. First, the ability of each real-time PCR assay to detect and quantify mixed infections was determined in vitro by testing mixtures of plasmids containing Mhm and Mhf 16S rDNA with each assay. Subsequently, 4 specific pathogen-free (SPF) cats, 2 of which were splenectomized, were inoculated with blood from a cat infected with both Mhm and Mhf. Sixteen blood samples were then collected from each cat over a 55-day period. Each of the 64 postinoculation samples was tested using both conventional polymerase chain reaction (cPCR) and real-time PCR for the 16S rRNA gene of each organism. When applied to mixtures of plasmid DNA from each species, the results of quantitation with each of the real-time PCR assays approximately reflected the number of plasmid copies present. Forty-nine of 64 post-inoculation samples (77{\%}) were positive using both cPCR and real-time PCR, 4 (6{\%}) were positive using cPCR only, and 3 (5{\%}) were positive using real-time PCR only. Both organisms were detected in 23 samples using real-time PCR. Mixed infections were not detected using cPCR. The size of the corresponding cPCR products suggested infection with Mhm in 4 and Mhf in 18 of these samples. The use of multiple separate real-time PCR assays rather than cPCR alone should thus be considered for epidemiologic studies of hemoplasmosis in cats.",
keywords = "Anemia, Haemobartonella, hemoplasma, quantitative PCR, splenectomy",
author = "Sykes, {Jane E} and Drazenovich, {Nicole L.} and Kyles, {Andrew E.} and Ball, {Louise M.} and Leutenegger, {Christian M.}",
year = "2007",
doi = "10.1177/104063870701900304",
language = "English (US)",
volume = "19",
pages = "250--255",
journal = "Journal of Veterinary Diagnostic Investigation",
issn = "1040-6387",
publisher = "American Association of Veterinary Laboratory Diagnosticians",
number = "3",

}

TY - JOUR

T1 - Detection of Mixed Infections with "Candidatus Mycoplasma Haemominutum" and Mycoplasma Haemofelis Using Real-Time TaqMan Polymerase Chain Reaction

AU - Sykes, Jane E

AU - Drazenovich, Nicole L.

AU - Kyles, Andrew E.

AU - Ball, Louise M.

AU - Leutenegger, Christian M.

PY - 2007

Y1 - 2007

N2 - The objective of this study was to evaluate the use of real-time TaqMan PCR assays for detection of coinfections with "Candidatus Mycoplasma haemominutum" (Mhm), and Mycoplasma haemofelis (Mhf), in vitro and over time in experimentally infected cats. First, the ability of each real-time PCR assay to detect and quantify mixed infections was determined in vitro by testing mixtures of plasmids containing Mhm and Mhf 16S rDNA with each assay. Subsequently, 4 specific pathogen-free (SPF) cats, 2 of which were splenectomized, were inoculated with blood from a cat infected with both Mhm and Mhf. Sixteen blood samples were then collected from each cat over a 55-day period. Each of the 64 postinoculation samples was tested using both conventional polymerase chain reaction (cPCR) and real-time PCR for the 16S rRNA gene of each organism. When applied to mixtures of plasmid DNA from each species, the results of quantitation with each of the real-time PCR assays approximately reflected the number of plasmid copies present. Forty-nine of 64 post-inoculation samples (77%) were positive using both cPCR and real-time PCR, 4 (6%) were positive using cPCR only, and 3 (5%) were positive using real-time PCR only. Both organisms were detected in 23 samples using real-time PCR. Mixed infections were not detected using cPCR. The size of the corresponding cPCR products suggested infection with Mhm in 4 and Mhf in 18 of these samples. The use of multiple separate real-time PCR assays rather than cPCR alone should thus be considered for epidemiologic studies of hemoplasmosis in cats.

AB - The objective of this study was to evaluate the use of real-time TaqMan PCR assays for detection of coinfections with "Candidatus Mycoplasma haemominutum" (Mhm), and Mycoplasma haemofelis (Mhf), in vitro and over time in experimentally infected cats. First, the ability of each real-time PCR assay to detect and quantify mixed infections was determined in vitro by testing mixtures of plasmids containing Mhm and Mhf 16S rDNA with each assay. Subsequently, 4 specific pathogen-free (SPF) cats, 2 of which were splenectomized, were inoculated with blood from a cat infected with both Mhm and Mhf. Sixteen blood samples were then collected from each cat over a 55-day period. Each of the 64 postinoculation samples was tested using both conventional polymerase chain reaction (cPCR) and real-time PCR for the 16S rRNA gene of each organism. When applied to mixtures of plasmid DNA from each species, the results of quantitation with each of the real-time PCR assays approximately reflected the number of plasmid copies present. Forty-nine of 64 post-inoculation samples (77%) were positive using both cPCR and real-time PCR, 4 (6%) were positive using cPCR only, and 3 (5%) were positive using real-time PCR only. Both organisms were detected in 23 samples using real-time PCR. Mixed infections were not detected using cPCR. The size of the corresponding cPCR products suggested infection with Mhm in 4 and Mhf in 18 of these samples. The use of multiple separate real-time PCR assays rather than cPCR alone should thus be considered for epidemiologic studies of hemoplasmosis in cats.

KW - Anemia

KW - Haemobartonella

KW - hemoplasma

KW - quantitative PCR

KW - splenectomy

UR - http://www.scopus.com/inward/record.url?scp=34250674890&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34250674890&partnerID=8YFLogxK

U2 - 10.1177/104063870701900304

DO - 10.1177/104063870701900304

M3 - Article

C2 - 17459853

AN - SCOPUS:34250674890

VL - 19

SP - 250

EP - 255

JO - Journal of Veterinary Diagnostic Investigation

JF - Journal of Veterinary Diagnostic Investigation

SN - 1040-6387

IS - 3

ER -