Evaluation of novel multiplex qPCR assays for diagnosis of pathogens associated with the bovine respiratory disease complex.

Bovine respiratory disease complex is the most common disease requiring the use of antimicrobials in industrial calf production worldwide. Pathogenic bacteria (Mannheimia haemolytica (Mh), Pasteurella multocida (Pm), Histophilus somni (Hs), and Mycoplasma bovis) and a range of viruses (bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza virus type 3, bovine viral diarrhea virus and bovine herpesvirus type 1) are associated with this complex. As most of these pathogens can be present in healthy and diseased calves, simple detection of their presence in diseased calves carries low predictive value. In other multi-agent diseases of livestock, quantification of pathogens has added substantially to the predictive value of microbiological diagnosis. The aim of this study was to evaluate the ability of two recently developed quantitative PCR (qPCR) kits (Pneumo4B and Pneumo4V) to detect and quantify these bacterial and viral pathogens, respectively. Test efficiencies of the qPCR assays, based on nucleic acid dilution series of target bacteria and viruses, were 93-106% and 91-104%, respectively, with assay detection limits of 10-50 copies of nucleic acids. All 44 strains of target bacteria were correctly identified, with no false positive reactions in 135strains of non-target bacterial species. Based on standard curves of log10 CFU versus cycle threshold (Ct) values, quantification was possible over a 5-log range of bacteria. In 92 tracheal aspirate samples, the kappa values for agreement between Pneumo4B and bacterial culture were 0.64-0.84 for Mh, Pm and Hs. In an additional 84 tracheal aspirates, agreement between Pneumo4B or Pneumo 4V and certified diagnostic qPCR assays was moderate (0.57) for M. bovis and high (0.71-0.90) for viral pathogens. Thus Pneumo4 kits specifically detected and quantified the relevant pathogens.

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.

Within-herd prevalence thresholds for herd-level detection of mastitis pathogens using multiplex real-time PCR in bulk tank milk samples.

The objective of the study was to assess the value of quantitative multiplex real-time PCR examination of bulk tank milk samples for bovine mastitis pathogens as a tool for herd level diagnosis. Using a logistic regression model, this study is aimed at calculating the threshold level of the apparent within-herd prevalence as determined by quarter milk sample cultivation of all lactating cows, thus allowing the detection of a herd positive for a specific pathogen within certain probability levels. A total of 6,335 quarter milk samples were collected and cultured from 1,615 cows on 51 farms in Germany. Bulk tank milk samples were taken from each farm and tested by bacterial culture as well as the commercial PCR assay Mastit 4A (DNA Diagnostic A/S, Risskov, Denmark) identifying Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus agalactiae, and Streptococcus uberis. In addition, PCR was performed on pooled herd milk samples containing milk aliquots from all lactating cows in each of the 51 herds. Only 1 out of the 51 herds was found PCR positive for Streptococcus agalactiae in bulk tank and pooled herd milk samples, and cultured quarter milk samples. Spearman's rank correlations between the cycle threshold value of bulk tank milk PCR and the apparent within-herd prevalence were calculated in regard to Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis. For these pathogens, significant correlations were found. If 1 bulk tank milk sample per herd was tested, the estimated within-herd prevalence thresholds for 90% probability of detection were 27.6% for Staphylococcus aureus, 9.2% for Streptococcus dysgalactiae, and 13.8% for Streptococcus uberis on the cow level. On the quarter level, the within-herd prevalence had to be at least 32.6% for Staphylococcus aureus, 1.7% for Streptococcus dysgalactiae, and 4.3% for Streptococcus uberis to detect a herd as positive using a single bulk milk sample. The results indicate that mastitis pathogens in bulk tank milk can be identified by the applied PCR assay. Bulk tank milk examination is not a reliable tool for the identification of the named pathogens by single testing, but might be a valuable monitoring tool when used frequently with repeated testing. Furthermore, this approach could be a useful monitoring tool for detecting new pathogen occurrence in the herd.

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.

Efficacy of enrofloxacin for the treatment of acute clinical mastitis caused by Escherichia coli in dairy cows.

Evidence for the efficacy of antimicrobial treatments in Escherichia coli mastitis is limited. The aim of this double-blinded field trial was to investigate the efficacy of enrofloxacin compared with placebo, with a special focus on survival, in dairy cows with acute clinical mastitis caused by E. coli. Dairy cows (n=116) with acute clinical mastitis were included in the study. A clinical examination was performed and a milk sample from the affected udder quarter was collected for investigation of somatic cell count (SCC) and bacteriology on the first day of treatment (day 0) and at day 3 (clinical examination only), day 22 and day 28. Data regarding culled cows, SCC and daily milk yield were retrieved from monthly milk recording each month until 180 days after treatment. All cows were treated with either enrofloxacin or placebo once a day for three days, starting at day 0. After culturing, 56 cows with confirmed E. coli mastitis remained in the study. Nine (16 per cent) of them died within the first week. Enrofloxacin-treated cows had lower SCC compared with placebo-treated cows at first monthly milk recordings after being treated for mastitis. Treatment with enrofloxacin did not result in a higher probability of survival compared with placebo.

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.

Quality of bulk tank milk samples from Danish dairy herds based on real-time polymerase chain reaction identification of mastitis pathogens.

Results of a commercial real-time PCR analysis for 11 mastitis pathogens from bulk tank milk (BTM) samples from all 4,258 Danish dairy herds in November 2009 to January 2010 were compared with somatic cell count (SCC) and total bacteria count (TBC) estimates in BTM. For Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis, a low real-time PCR cycle threshold (Ct) value (corresponding to high bacterial DNA quantity) was correlated with higher SCC and higher TBC. For Staphylococcus aureus, low Ct values were correlated only with higher SCC. For the environmental mastitis pathogens Klebsiella spp., Enterococcus spp., and Escherichia coli, low Ct values had a correlation with higher TBC. Staphylococcus spp. were found in the BTM from all herds, Strep. uberis in 95%, Staph. aureus in 91%, and Strep. dysgalactiae in 86%, whereas E. coli, Klebsiella, and Strep. agalactiae were found in 61, 13, and 7% of the herds. It is concluded that the real-time PCR used provides results that are related to the milk quality in the herds. Real-time PCR can be used in the same way as culture for monitoring BTM samples, and is especially useful for bacteria with low prevalence (e.g., Strep. agalactiae).