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Real-time PCR of the mammalian hydroxymethylbilane synthase (HMBS) gene for analysis of flea (Ctenocephalides felis) feeding patterns on dogs.

Wang C, Mount J, Butler J, Gao D, Jung E, Blagburn BL, Kaltenboeck B - Parasit Vectors (2012)

Bottom Line: Sensitivity of the assay was established by limiting dilution and detection of single copies of HMBS DNA equivalent to 0.043 nL blood.Application of the assay indicated that after 15 minutes on a dog, male and female fleas had ingested low, but similar amounts of approximately 1.1. nL blood.The HMBS PCR method developed here offers the advantages of both exquisite sensitivity and specificity that make it superior to other approaches for quantification of blood ingested by fleas.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849-5519, USA.

ABSTRACT

Background: Precise data on quantitative kinetics of blood feeding of fleas, particularly immediately after contact with the host, are essential for understanding dynamics of flea-borne disease transmission and for evaluating flea control strategies. Standard methods used are inadequate for studies that simulate early events after real-life flea access to the host.

Methods: Here, we developed a novel quantitative polymerase chain reaction targeting mammalian DNA within fleas to quantify blood consumption with high sensitivity and specificity. We used primers and fluorescent probes that amplify the hydroxymethylbilane synthase (HMBS) gene, an evolutionary divergent gene that is unlikely to be detected in insects by mammalian-specific primers and probes. To validate this assay, fleas were placed on dogs, allowed to distribute in the hair, and removed at specific time points with single-use combs. Fleas were then immediately homogenized by vigorous shaking with ceramic beads in guanidinium-based DNA preservation buffer for DNA extraction.

Results: The specificity of this assay was ascertained by amplification of canine, feline and equine blood with differential product melting temperatures (Tm), and lack of amplification of bovine and porcine blood and of adult fleas reared from larvae fed with bovine blood. Sensitivity of the assay was established by limiting dilution and detection of single copies of HMBS DNA equivalent to 0.043 nL blood. Application of the assay indicated that after 15 minutes on a dog, male and female fleas had ingested low, but similar amounts of approximately 1.1. nL blood. Saturation uptake of 118 and 100 nL blood per flea was found at 30 and 60 min on the dog, respectively.

Conclusions: The HMBS PCR method developed here offers the advantages of both exquisite sensitivity and specificity that make it superior to other approaches for quantification of blood ingested by fleas. The capability to detect minute quantities of blood in single fleas, particularly immediately after colonization of the host, will provide a superior tool for studying flea-host interactions, flea-borne disease transmission, and flea control strategies.

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Alignment of HMBS amplification targets of Canis lupus familiaris, Felis catus, Equus caballus, Bos taurus, and jewel wasp Nasonia vitripennis. Dots indicate nucleotides identical to the canine HMBS sequence, and dashes represent nucleotide deletions. Primers and probes are shown by boxes. The upstream primer is used as shown while the downstream primer and probes are applied as reverse complement (antisense) oligonucleotides. The probes were designed for discrimination of the homologous sequences of different animal species. K represents G or T, and R stands for A or G.
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Figure 1: Alignment of HMBS amplification targets of Canis lupus familiaris, Felis catus, Equus caballus, Bos taurus, and jewel wasp Nasonia vitripennis. Dots indicate nucleotides identical to the canine HMBS sequence, and dashes represent nucleotide deletions. Primers and probes are shown by boxes. The upstream primer is used as shown while the downstream primer and probes are applied as reverse complement (antisense) oligonucleotides. The probes were designed for discrimination of the homologous sequences of different animal species. K represents G or T, and R stands for A or G.

Mentions: Nucleotide sequences of the HMBS genes of Canis lupus familiaris (NC_006587.2, whole gene region: 17774346-17781513), Felis catus (AANG01077049, amplification product region: 15323-15521), Equus caballus (AAWR02020903.1, amplification product region: 93984-94182), Bos taurus (AAFC03046256.1, region homologous to amplification product: 61960-61765), and the jewel wasp Nasonia vitripennis (AAZX01001902.1, region homologous to amplification product: 13757- 13550) were obtained from GenBank. Primers and probes were designed by use of the Vector NTI software (Invitrogen Corporation, Carlsbad, CA, USA) and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The HMBS nucleotide target sequences and positions of primers and probes are shown in Figure 1. The HMBS upstream and downstream primers were placed on introns 4 and 5 (CaFeHMBSUP, 5'-TTCCTTCCCCCAAAAGATTCACTCTG-3'; and CaFeHMBSDN, 5'-TGAAGYCCCMCAGTCTAGCTGATAT-3'), and the HMBS probes were placed on exon 5. The fluorescein probe (CaFeHMBSFLU, 5'-CTTTTCCAGCGCGTGTTCCAGCTC-6-FAM-3') was 3'-labeled with carboxyfluorescein (6-FAM). This probe was used unpurified and acts as a fluorescence resonance energy (FRET) donor probe excited by 488 nm light. The LightCycler Red 640 probe (CaFeHMBSLCR640, 5'-LCRed640-TTGGTAAACAGGCTCTTCTCGCCAA-Phosphate-3') was HPLC-purified and used as FRET acceptor probe, emitting 640 nm fluorescence following excitation by physical proximity to 6-FAM. Primers were designed to amplify DNA of mammalian host species of fleas, including prominently dogs and cats, but not cattle, the blood of which is used for maintenance of flea colonies, or arthropods. The fluorescein probe is also designed for maximum discrimination between these species, while the LightCycler Red probe hybridizes to the known relevant mammalian homologs, but not to arthropod sequences.


Real-time PCR of the mammalian hydroxymethylbilane synthase (HMBS) gene for analysis of flea (Ctenocephalides felis) feeding patterns on dogs.

Wang C, Mount J, Butler J, Gao D, Jung E, Blagburn BL, Kaltenboeck B - Parasit Vectors (2012)

Alignment of HMBS amplification targets of Canis lupus familiaris, Felis catus, Equus caballus, Bos taurus, and jewel wasp Nasonia vitripennis. Dots indicate nucleotides identical to the canine HMBS sequence, and dashes represent nucleotide deletions. Primers and probes are shown by boxes. The upstream primer is used as shown while the downstream primer and probes are applied as reverse complement (antisense) oligonucleotides. The probes were designed for discrimination of the homologous sequences of different animal species. K represents G or T, and R stands for A or G.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3275464&req=5

Figure 1: Alignment of HMBS amplification targets of Canis lupus familiaris, Felis catus, Equus caballus, Bos taurus, and jewel wasp Nasonia vitripennis. Dots indicate nucleotides identical to the canine HMBS sequence, and dashes represent nucleotide deletions. Primers and probes are shown by boxes. The upstream primer is used as shown while the downstream primer and probes are applied as reverse complement (antisense) oligonucleotides. The probes were designed for discrimination of the homologous sequences of different animal species. K represents G or T, and R stands for A or G.
Mentions: Nucleotide sequences of the HMBS genes of Canis lupus familiaris (NC_006587.2, whole gene region: 17774346-17781513), Felis catus (AANG01077049, amplification product region: 15323-15521), Equus caballus (AAWR02020903.1, amplification product region: 93984-94182), Bos taurus (AAFC03046256.1, region homologous to amplification product: 61960-61765), and the jewel wasp Nasonia vitripennis (AAZX01001902.1, region homologous to amplification product: 13757- 13550) were obtained from GenBank. Primers and probes were designed by use of the Vector NTI software (Invitrogen Corporation, Carlsbad, CA, USA) and synthesized by Integrated DNA Technologies (Coralville, IA, USA). The HMBS nucleotide target sequences and positions of primers and probes are shown in Figure 1. The HMBS upstream and downstream primers were placed on introns 4 and 5 (CaFeHMBSUP, 5'-TTCCTTCCCCCAAAAGATTCACTCTG-3'; and CaFeHMBSDN, 5'-TGAAGYCCCMCAGTCTAGCTGATAT-3'), and the HMBS probes were placed on exon 5. The fluorescein probe (CaFeHMBSFLU, 5'-CTTTTCCAGCGCGTGTTCCAGCTC-6-FAM-3') was 3'-labeled with carboxyfluorescein (6-FAM). This probe was used unpurified and acts as a fluorescence resonance energy (FRET) donor probe excited by 488 nm light. The LightCycler Red 640 probe (CaFeHMBSLCR640, 5'-LCRed640-TTGGTAAACAGGCTCTTCTCGCCAA-Phosphate-3') was HPLC-purified and used as FRET acceptor probe, emitting 640 nm fluorescence following excitation by physical proximity to 6-FAM. Primers were designed to amplify DNA of mammalian host species of fleas, including prominently dogs and cats, but not cattle, the blood of which is used for maintenance of flea colonies, or arthropods. The fluorescein probe is also designed for maximum discrimination between these species, while the LightCycler Red probe hybridizes to the known relevant mammalian homologs, but not to arthropod sequences.

Bottom Line: Sensitivity of the assay was established by limiting dilution and detection of single copies of HMBS DNA equivalent to 0.043 nL blood.Application of the assay indicated that after 15 minutes on a dog, male and female fleas had ingested low, but similar amounts of approximately 1.1. nL blood.The HMBS PCR method developed here offers the advantages of both exquisite sensitivity and specificity that make it superior to other approaches for quantification of blood ingested by fleas.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849-5519, USA.

ABSTRACT

Background: Precise data on quantitative kinetics of blood feeding of fleas, particularly immediately after contact with the host, are essential for understanding dynamics of flea-borne disease transmission and for evaluating flea control strategies. Standard methods used are inadequate for studies that simulate early events after real-life flea access to the host.

Methods: Here, we developed a novel quantitative polymerase chain reaction targeting mammalian DNA within fleas to quantify blood consumption with high sensitivity and specificity. We used primers and fluorescent probes that amplify the hydroxymethylbilane synthase (HMBS) gene, an evolutionary divergent gene that is unlikely to be detected in insects by mammalian-specific primers and probes. To validate this assay, fleas were placed on dogs, allowed to distribute in the hair, and removed at specific time points with single-use combs. Fleas were then immediately homogenized by vigorous shaking with ceramic beads in guanidinium-based DNA preservation buffer for DNA extraction.

Results: The specificity of this assay was ascertained by amplification of canine, feline and equine blood with differential product melting temperatures (Tm), and lack of amplification of bovine and porcine blood and of adult fleas reared from larvae fed with bovine blood. Sensitivity of the assay was established by limiting dilution and detection of single copies of HMBS DNA equivalent to 0.043 nL blood. Application of the assay indicated that after 15 minutes on a dog, male and female fleas had ingested low, but similar amounts of approximately 1.1. nL blood. Saturation uptake of 118 and 100 nL blood per flea was found at 30 and 60 min on the dog, respectively.

Conclusions: The HMBS PCR method developed here offers the advantages of both exquisite sensitivity and specificity that make it superior to other approaches for quantification of blood ingested by fleas. The capability to detect minute quantities of blood in single fleas, particularly immediately after colonization of the host, will provide a superior tool for studying flea-host interactions, flea-borne disease transmission, and flea control strategies.

Show MeSH
Related in: MedlinePlus