Direct quantification of Campylobacter jejuni and Campylobacter lanienae in feces of cattle by real-time quantitative PCR

Abstract

Campylobacter species are fastidious to culture, and the ability to directly quantify biomass in microbiologically complex substrates using real-time quantitative (RTQ) PCR may enhance our understanding of their biology and facilitate the development of efficacious mitigation strategies. This study reports the use of nested RTQ-PCR to directly quantify Campylobacter jejuni and Campylobacter lanienae in cattle feces. For C. jejuni, the single-copy mapA gene was selected. For C. lanienae, the three-copy 16S rRNA gene was targeted. RTQ-PCR primers were tested alone or they were nested with species-specific primers, and amplification products were detected using the intercalating dye SYBR Green. Nesting did not increase the specificity or sensitivity of C. jejuni quantification, and the limit of quantification was 19 to 25 genome copies ( approximately 3 x 10(3) CFU/g of feces). In contrast, nested RTQ-PCR was necessary to confer specificity on C. lanienae by targeting the 16S rRNA gene. The limit of quantification was 1.8 genome copies ( approximately 250 CFU/g of feces), and there was no discernible difference between the two C. lanienae secondary primer sets evaluated. Detection and quantification of C. jejuni in naturally infested cattle feces by RTQ-PCR were comparable to the results of culture-based methods. In contrast, culturing did not detect C. lanienae in 6 of 10 fecal samples positive for the bacterium and substantially underestimated cell densities relative to nested RTQ-PCR. The results of this study illustrate that RTQ-PCR can be used to directly quantify campylobacters, including very fastidious species, in a microbiologically and chemically complex substrate. Furthermore, targeting of a multicopy universal gene provided highly sensitive quantification of C. lanienae, but nested RTQ-PCR was necessary to confer specificity. This method will facilitate subsequent studies to elucidate the impact of this group of bacteria within the gastrointestinal tracts of livestock and studies of the factors that influence colonization success and shedding.

FIG. 1.

Detection of C. jejuni in inoculated cattle feces by targeting the single-copy mapA gene with nested (solid symbols) and nonnested (open symbols) RTQ-PCRs. The log linear equations to describe the relationships are as follows: nested, y = −1.49 + 0.85x, r² = 0.99; nonnested, y = −1.85 + 0.91x, r² = 0.99. The error bars representing standard errors of the mean (n = 3) are within the symbols. The dotted lines represent 95% confidence intervals.

FIG. 2.

Threshold values for C. jejuni in inoculated cattle feces by targeting the single-copy mapA gene for nested (solid symbols) and nonnested (open symbols) RTQ-PCRs. The linear equations to describe the relationships are as follows: nested, y = 23.22 − 3.80x, r² = 0.97; nonnested, y = 41.74 − 3.58x, r² = 0.92. The dotted lines represent 95% confidence intervals.

FIG. 3.

Quantification of C. lanienae in inoculated cattle feces by targeting the three-copy 16S rRNA gene with nested (solid symbols) and nonnested (open symbols) RTQ-PCRs. (A) Primer set 1, where the log quadratic polynomial equations to describe the relationships are as follows: nested, y = 2.23 − 0.20x + 0.087x², r² = 0.989; nonnested, y = −1.44 + 0.49x + 0.059x², r² = 0.996. (B) Primer set 2, where the log quadratic polynomial equations to describe the relationships are as follows: nested, y = −1.58 + 0.66x + 0.032x², r² = 0.997; nonnested, y = 1.20 + 0.053x + 0.077x², r² = 0.986. The error bars associated with the symbols represent standard errors of the mean (n = 3), and bars that are not visible are within the symbols. The dotted lines represent 95% confidence intervals.

FIG. 4.

Populations of Campylobacter species in uninoculated cattle feces as determined by culture-based enumeration. (A) CCDA containing selective supplement SR115E at 37°C. (B) Karmali agar with selective supplement CM935 at 40°C. Identifications were made using colony PCR (8), and the asterisks indicate missing data.

FIG. 5.

Quantification comparison of C. jejuni and C. lanienae in uninoculated beef cattle feces (20 animals) among culture-based isolation on Karmali agar containing selective supplement CM935 at 40°C (CFU-Karmali), nested RTQ-PCR, and nonnested RTQ-PCR. (A) C. jejuni by targeting the mapA gene. (B) C. lanienae by targeting the 16S rRNA gene with primer set 1. (C) C. lanienae by targeting the 16S rRNA gene with primer set 2. With the exception of bars labeled 1 (n = 1), the error bars represent standard deviations (n = 2). *, detection disparity between culture-based isolation and nested RTQ-PCR; +, detection disparity between culture-based isolation and nonnested RTQ-PCR.