Diagnostic accuracy of two DNA-based molecular assays for detection of porcine circovirus 3 in swine population using Bayesian latent class analysis.

Molecular-based tools sometimes are the only laboratory techniques available to detect a recently discovered agent, and their validation without the existence of previously described 'gold standard' methods poses a challenge for the diagnosticians. A good example within this scenario is the recently described porcine circovirus 3 (PCV-3) in the swine population worldwide, from which only few PCR methods have been described. Therefore, the primary objective of this study was to estimate the diagnostic accuracy of a direct PCR (dPCR) and a real-time qPCR (qPCR) for detection of PCV-3 in Italian swine population. Bayesian latent class analysis approach was used to rigorously assess their features and applicability in routine diagnostic activity. Data on dPCR and qPCR were available from 116 domestic pigs, which were randomly selected from 55 farms located at different regions in Northern Italy. The sensitivity (Se) estimates of dPCR (94%; posterior credible interval (PCI%) 84-100) and qPCR (96%; PCI% 90-100) were high and similar. The estimated specificity (Sp) of both dPCR and qPCR assays was around 97%. dPCR and qPCR assays showed a high and comparable Se and Sp estimates for the detection of PCV-3 in Italian swine population. SIGNIFICANCE AND IMPACT OF THE STUDY: The continuous discovery of new pathogens poses a challenge in the development and evaluation of adequate diagnostic tools. In fact, since molecular-based tools sometimes are the only available laboratory techniques, it is typically difficult to evaluate their diagnostic performances in the absence of a gold standard. The present study assesses this issue, demonstrating the excellent performances of two PCR-based assays for porcine circovirus 3 (PCV-3) detection using a Bayesian latent class analysis approach. Therefore, the molecular tests evaluated under this study constitute reliable tools for the routine diagnosis and surveillance programs of PCV-3 circulating in swine populations.

Bayesian estimation of qPCR and bacterial culture accuracy for detection of bovine coagulase-negative staphylococci from milk and teat apex at different test cut-off points.

AIM: To primarily estimate the sensitivity (Se) and specificity (Sp) of the commercially available Mastit4 quantitative PCR (qPCR) assay and bacterial culture (BC) for diagnosis of intramammary infections (IMI) and teat apex colonization (TAC) with coagulase-negative staphylococci (CNS) at different cut-offs for qPCR cycle threshold values using Bayesian latent class analysis. A secondary objective was to evaluate two cut-offs of BC for diagnosis of IMI and TAC with CNS. METHODS AND RESULTS: We randomly selected 13-20 cows with subclinical mastitis from eight dairy herds. Teat skin samples and aseptically collected foremilk samples were collected from the right hindquarters (n = 149) for BC and qPCR analysis. The Se of qPCR was always higher than BCSe in diagnosis of IMI, however; the Sp of BC was higher than qPCRSp . BCSe and BCSp showed no substantial difference between the tested BC cut-offs. In contrast to IMI, estimates of BC and qPCR in diagnosing TAC were different. BCSe was higher than qPCRSe at all tested cut-offs, however; qPCRSp was higher than BCSp . CONCLUSION: The overall performance of qPCR is higher than BC in the diagnosis of IMI; however, the performance of BC is better than qPCR in diagnosis of TAC. The qPCR and BC are valid diagnostics for bovine IMI with CNS. However, for TAC, both techniques require further investigation to reduce the uncertainty of the true status of the quarter and teat skin. SIGNIFICANCE AND IMPACT OF THE STUDY: We reported, for the first time, the diagnostic performance of new mastitis technology (Mastit4 PCR) and culture for detection of CNS in milk and nonmilk samples in dairy herds with automatic milking systems. Our findings will improve the interpretation of the test results of culture and qPCR assay and subsequently, will strengthen the control of IMI with CNS in dairy cows.

Molecular epidemiology and strain-specific characteristics of Streptococcus agalactiae at the herd and cow level.

Host-adaptation of Streptococcus agalactiae subpopulations has been described whereby strains that are commonly associated with asymptomatic carriage or disease in people differ phenotypically and genotypically from those causing mastitis in dairy cattle. Based on multilocus sequence typing (MLST), the most common strains in dairy herds in Denmark belong to sequence types (ST) that are also frequently found in people. The aim of this study was to describe epidemiological and diagnostic characteristics of such strains in relation to bovine mastitis. Among 1,199 cattle from 6 herds, cow-level prevalence of S. agalactiae was estimated to be 27.4% based on PCR and 7.8% based on bacteriological culture. Quarter-level prevalence was estimated at 2.8% based on bacteriological culture. Per herd, between 2 and 26 isolates were characterized by pulsed-field gel electrophoresis (PFGE) and MLST. Within each herd, a single PFGE type and ST predominated, consistent with a contagious mode of transmission or point source infection within herds. Evidence of within-herd evolution of S. agalactiae was detected with both typing methods, although ST belonged to a single clonal complex (CC) per herd. Detection of CC23 (3 herds) was associated with significantly lower approximate count (colony-forming units) at the quarter level and significantly lower cycle threshold value at the cow level than detection of CC1 (2 herds) or CC19 (1 herd), indicating a lower bacterial load in CC23 infections. Median values for the number of infected quarters and somatic cell count (SCC) were numerically but not significantly lower for cows infected with CC23 than for cows with CC1 or CC19. For all CC, an SCC threshold of 200,000 cells/mL was an unreliable indicator of infection status, and prescreening of animals based on SCC as part of S. agalactiae detection and eradication campaigns should be discouraged.

Effect of presampling procedures on real-time PCR used for diagnosis of intramammary infections with Staphylococcus aureus in dairy cows at routine milk recordings.

Aseptic procedures for milk sample collection are considered crucial for bacterial culture to avoid misdiagnosis and subsequently unnecessary treatment or culling. The objective of this field study was to investigate the effect of presampling procedures on the PCR-positivity at cycle threshold value ≤ 37 of real-time PCR assay to detect Staphylococcus aureus from composite milk samples at routine milk recordings while accounting for known cow-level risk factors. A total of 1,199 dairy cows from 6 herds with conventional milking parlors were sampled and tested by PCR in 2011. Following the farmers' routine premilking preparations, 624 cows of the 1,199 cows were randomly selected for bacterial culture preceded by presampling procedures. These procedures were: cleaning of udder teats, removing the first streams of milk, and 70% alcohol teat disinfection. Data on parity, somatic cell counts, days in milk, daily milk yield, fat %, and protein % were extracted from the Danish Cattle Database, whereas energy-corrected milk was calculated based on the latter 3. The within-herd prevalence of intramammary infections with Staph. aureus was 31%, ranging from 16 to 48% in the 6 herds. Univariable analysis showed that the presampling procedures, somatic cell counts, energy-corrected milk, and days in milk were significantly associated with PCR-positivity, whereas parity was not significant. A multivariable model with herd as random effect showed that presampling procedures decreased the chance of being PCR-positive to 0.75 (95% CI; 0.58-0.97) compared with cows where the presampling procedures were not carried out. In conclusion, presampling procedures decreased the cow's chance of being PCR-positive to Staph. aureus. Presampling procedures appeared to improve the specificity of PCR for detection of Staph. aureus by reducing false positives through destruction of Staph. aureus bacteria colonizing or contaminating the teat skin, orifice, and canal. Random herd effects accounted for only 8.9%, indicating that the cluster effect due to herd management on the PCR positivity to Staph. aureus was virtually negligible.

Specific PCR-based assays for the identification of Fasciola species: their development, evaluation and potential usefulness in prevalence surveys.

Among the helminths infecting ruminants in China are three taxa belonging to the genus Fasciola: F. hepatica, F. gigantica and the so-called 'intermediate form' that appears to lie between these two species. Based on the sequences of the second internal-transcribed spacers (ITS-2) within the parasites' nuclear ribosomal DNA (rDNA), a pair of primers (DSJf/DSJ3) specific for F. hepatica and a pair (DSJf/DSJ4) specific for F. gigantica were designed and used to develop PCR-based assays. These assays allowed the identification and differentiation of F. hepatica, F. gigantica and the 'intermediate' Fasciola, with no amplicons produced from heterologous DNA samples. The results of sequencing confirmed the species-specific identity of the amplified products. The assays showed good sensitivity, giving positive results with as little as 0.11 ng of F. hepatica DNA and 0.35 ng of F. gigantica DNA. This meant that the DNA from a single Fasciola egg or a single infected snail was sufficient for identification of the Fasciola taxon. The developed PCR assays could provide useful tools for the detection, identification and epidemiological investigation of Fasciola infection in humans, other mammals and snails.