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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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Related in: MedlinePlus

Quantification accuracy of canine blood ingestion by fleas. HMBS copies were quantified for individual fleas fed only bovine blood that were collected directly from the flea colony followed immediately by homogenization and nucleic acid extraction (flea colony; n = 10 fleas). Another group contained fleas that were immediately removed from the dog and DNA was immediately extracted (collection control; n = 16). For evaluation of HMBS DNA recovery, fleas collected after 4 hours on the dog were either stored for 7 days at room temperature in RNA/DNA Stabilization buffer followed by homogenization (RT - homogenize; n = 10) or were homogenized immediately after collection followed by 7-day RT storage (homogenize - RT; n = 10). HMBS amplification was not observed in fleas from the colony, while fleas in the collection control group did contain a small but significant amount of HMBS targets indicating a background level of contamination with canine DNA by contact of the fleas with the dog. Fleas of the homogenize-RT group carried significantly higher HMBS copy numbers than RT-homogenize fleas (P = 0.006) indicating incomplete preservation of DNA in non-homogenized fleas. Fleas of the homogenize-RT group (the final collection approach) contained highly significantly more HMBS copies than those of the collection control group (P = 0.001), indicating a significant amount of blood feeding. Thick bars indicate the mean HMBS, and error bars indicate minimum/maximum copies per 10 fleas.
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Figure 3: Quantification accuracy of canine blood ingestion by fleas. HMBS copies were quantified for individual fleas fed only bovine blood that were collected directly from the flea colony followed immediately by homogenization and nucleic acid extraction (flea colony; n = 10 fleas). Another group contained fleas that were immediately removed from the dog and DNA was immediately extracted (collection control; n = 16). For evaluation of HMBS DNA recovery, fleas collected after 4 hours on the dog were either stored for 7 days at room temperature in RNA/DNA Stabilization buffer followed by homogenization (RT - homogenize; n = 10) or were homogenized immediately after collection followed by 7-day RT storage (homogenize - RT; n = 10). HMBS amplification was not observed in fleas from the colony, while fleas in the collection control group did contain a small but significant amount of HMBS targets indicating a background level of contamination with canine DNA by contact of the fleas with the dog. Fleas of the homogenize-RT group carried significantly higher HMBS copy numbers than RT-homogenize fleas (P = 0.006) indicating incomplete preservation of DNA in non-homogenized fleas. Fleas of the homogenize-RT group (the final collection approach) contained highly significantly more HMBS copies than those of the collection control group (P = 0.001), indicating a significant amount of blood feeding. Thick bars indicate the mean HMBS, and error bars indicate minimum/maximum copies per 10 fleas.

Mentions: All statistical analyses were performed with the Statistica 7.1 software package (StatSoft, Inc., Tulsa, OK, USA). The background HMBS copies (31.5 copies/flea, or 315 copies/pool of 10 fleas) were subtracted from the original HMBS PCR data, and the data were logarithmically transformed. The Mann-Whitney U test was used to analyze HMBS copies in determination of quantification accuracy of canine blood ingestion by fleas (Figure 3) and of the ingestion of canine blood by individual male and female fleas (Figure 4). PCR data of the time-course of canine blood ingestion were analyzed by one-way ANOVA and Tukey HSD tests (Figure 5). Differences at P ≤ 0.05 were considered significant.


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)

Quantification accuracy of canine blood ingestion by fleas. HMBS copies were quantified for individual fleas fed only bovine blood that were collected directly from the flea colony followed immediately by homogenization and nucleic acid extraction (flea colony; n = 10 fleas). Another group contained fleas that were immediately removed from the dog and DNA was immediately extracted (collection control; n = 16). For evaluation of HMBS DNA recovery, fleas collected after 4 hours on the dog were either stored for 7 days at room temperature in RNA/DNA Stabilization buffer followed by homogenization (RT - homogenize; n = 10) or were homogenized immediately after collection followed by 7-day RT storage (homogenize - RT; n = 10). HMBS amplification was not observed in fleas from the colony, while fleas in the collection control group did contain a small but significant amount of HMBS targets indicating a background level of contamination with canine DNA by contact of the fleas with the dog. Fleas of the homogenize-RT group carried significantly higher HMBS copy numbers than RT-homogenize fleas (P = 0.006) indicating incomplete preservation of DNA in non-homogenized fleas. Fleas of the homogenize-RT group (the final collection approach) contained highly significantly more HMBS copies than those of the collection control group (P = 0.001), indicating a significant amount of blood feeding. Thick bars indicate the mean HMBS, and error bars indicate minimum/maximum copies per 10 fleas.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Quantification accuracy of canine blood ingestion by fleas. HMBS copies were quantified for individual fleas fed only bovine blood that were collected directly from the flea colony followed immediately by homogenization and nucleic acid extraction (flea colony; n = 10 fleas). Another group contained fleas that were immediately removed from the dog and DNA was immediately extracted (collection control; n = 16). For evaluation of HMBS DNA recovery, fleas collected after 4 hours on the dog were either stored for 7 days at room temperature in RNA/DNA Stabilization buffer followed by homogenization (RT - homogenize; n = 10) or were homogenized immediately after collection followed by 7-day RT storage (homogenize - RT; n = 10). HMBS amplification was not observed in fleas from the colony, while fleas in the collection control group did contain a small but significant amount of HMBS targets indicating a background level of contamination with canine DNA by contact of the fleas with the dog. Fleas of the homogenize-RT group carried significantly higher HMBS copy numbers than RT-homogenize fleas (P = 0.006) indicating incomplete preservation of DNA in non-homogenized fleas. Fleas of the homogenize-RT group (the final collection approach) contained highly significantly more HMBS copies than those of the collection control group (P = 0.001), indicating a significant amount of blood feeding. Thick bars indicate the mean HMBS, and error bars indicate minimum/maximum copies per 10 fleas.
Mentions: All statistical analyses were performed with the Statistica 7.1 software package (StatSoft, Inc., Tulsa, OK, USA). The background HMBS copies (31.5 copies/flea, or 315 copies/pool of 10 fleas) were subtracted from the original HMBS PCR data, and the data were logarithmically transformed. The Mann-Whitney U test was used to analyze HMBS copies in determination of quantification accuracy of canine blood ingestion by fleas (Figure 3) and of the ingestion of canine blood by individual male and female fleas (Figure 4). PCR data of the time-course of canine blood ingestion were analyzed by one-way ANOVA and Tukey HSD tests (Figure 5). Differences at P ≤ 0.05 were considered significant.

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