Quantitative Detection of Chlamydia psittaci and C. pecorum by High-Sensitivity Real-Time PCR Reveals High Prevalence of Vaginal Infection in Cattle

Abstract

Bovine vaginal cytobrush specimens were analyzed for the presence of Chlamydia spp. by a high-sensitivity, high-specificity quantitative PCR. The 53% prevalence of low-level Chlamydia psittaci and C. pecorum genital infection detected in virgin heifers suggests predominantely extragenital transmission of Chlamydia in cattle and conforms to the high seroprevalence of anti-Chlamydia antibodies.Over the last 40 years, evidence has accumulated to suggest the ubiquitous presence of infections with intracellular bacteria of the genus Chlamydia in cattle and other livestock species. Despite some improvement in diagnostic techniques, our understanding about the prevalence and pathogenetic significance of these infections, succinctly reviewed by Shewen (11) in 1980, has not substantially changed since that time. In cattle, enzyme-linked immunosorbent assay examinations of sera for the antibody against Chlamydia psittaci suggest a high level of exposure to C. psittaci (6,8,10). The application of nested PCR to bovine clinical specimens substantiated such widespread, but mostly clinically inapparent, presumably low-level infections (3, 7), similar to the findings for human C. pneumoniae infections (1). However, due to high technical demands, these PCR methods were rarely transferred from research settings to systematic epidemiological investigations and diagnostic use. A simple, highly specific, fluorescentprobe-based single-tube LightCycler quantitative PCR (qPCR) platform was recently developed for the detection of Chlamydia DNA. This platform optimizes sample nucleic acid preservation, extraction, and recovery, as well as qPCR methodology, for maximum sensitivity in the detection of Chlamydia (2). This has opened the possibility for the sensitive routine diagnosis of chlamydial infection as well as for systematic epidemiological studies. Such investigations would benefit from both the high sensitivity and the ability to ascertain quantitative differences in chlamydial burdens between animals, which may be required to understand disease mechanisms. In this initial epidemiological application of the Chlamydia qPCR platform, we addressed the question of chlamydial infection of the bovine genital tract in animals that had not previously been exposed to the possibility of sexual transmission of chlamydiae. We report here a high prevalence of genital tract infection with C. psittaci and C. pecorum in clinically normal virgin cattle.A herd of 51 virgin Holstein heifers, 14 to 16 months old, was sampled four times at weekly intervals. These animals were clinically normal, but low-grade vaginitis was common.Vaginal cytobrush specimens (Histobrush; Fisher Scientific, Suwanee, Ga.) were obtained by 10-s rotation in the vaginal vestibulum, brushes were immediately transferred to 400 l of RNA-DNA stabilization reagent (Roche Applied Science, Indianapolis, Ind.) in a 1.5-ml screw-cap microcentrifuge tube, samples were stirred, and the brushes were cut off. Brushes were removed after a 5-min centrifugation at 3,000 ϫ g at room tempera...

High Prevalence of Natural Chlamydophila Species Infection in Calves

“…Recently, a highly sensitive real-time PCR method suitable for large-throughput routine detection, quantification, and differentiation of Chlamydophila DNA was established (7). By using vaginal cytobrush swabs of clinically normal virgin heifers, a 53% prevalence of C. abortus and C. pecorum infection was detected, supporting the notion of continuous low-level infection (6).…”

“…Quantitative standards used were 10 4 , 10 3 , 10 2 , and 10 copies of Chlamydophila abortus B577 and 10 copies of Chlamydophila pecorum LW613 DNA extracted from purified elementary body preparations by the High Pure method and quantified by a PicoGreen DNA fluorescence assay (Molecular Probes). Chlamydophila species were differentiated by melting curve analysis of the amplification products (6). C. abortus DNA in selected specimens was confirmed by C. psittaci B577 omp1 FRET-qPCR (7).…”

Development of a Chlamydophila psittaci species-specific and genotype-specific real-time PCR

“…felis [12,13,20] and Cp. pecorum [5]. The present study is the first to demonstrate the use of realtime PCR to specifically detect Cp.…”

Chlamydia pecorum

“…Molecular methods such as PCR followed by sequencing are more sensitive, specific, and less time-consuming methods for the detection and identification of these bacteria. 3,4,6,14,20,29 Some of the pathological findings in the fetuses examined in our study, such as cartilaginous metaplasia of the placenta in fetus A, and brachygnathia and palatoschisis in fetus B, cannot be explained by C. pecorum infection alone. Because brachygnathia has been described in aborted ruminants with orthobunyavirus infection, 15 we tested fetus B for the detection of bunyaviruses by PCR, with negative results.…”

“…C. pecorum subclinical intestinal infections have been reported in flocks with and without a history of abortion, with animals acting as asymptomatic carriers. 6,17,19,25,29 The enteric subtypes of C. pecorum are unlikely to be invasive and reach the placenta in pregnant animals when a well-balanced host-bacteria relationship is occurring. 26,32 Exceptionally, some carrier animals may develop severe disease as a result of stress, lapsing into clinical disease.…”