Diagnosing intramammary infections: evaluation of composite milk samples to detect intramammary infections.

Composite milk samples, in which milk from all 4 bovine quarters is collected in a single vial, are widely used in many developed dairy industries for detection of intramammary infections (IMI). These samples are more economical for use in culturing protocols than individual quarter samples, and may be useful when considering management options at the cow and herd level. The dilution effect may be problematic, however, resulting in lower sensitivity (Se) in IMI detection on composite samples. Relative Se and specificity (Sp) in composite samples have previously been described for some major pathogens, but because the causative organism for IMI is initially unknown, it is beneficial to investigate the reliability of composite samples for detection of all types of mastitis-causing bacteria. The Canadian Bovine Mastitis Research Network has a large data collection platform-the National Cohort of Dairy Farms-containing a vast amount of data on mastitis in Canada. These data have been used to further investigate the Se and Sp of composite samples in detecting IMI caused by specific mastitis pathogens. Milk samplings of selected cows before dry-off, after calving, and during lactation (n=48,835 samples) were employed to this end. Composite samples showed moderately high Se for Staphylococcus aureus (77.1%, 95% CI=73.3-80.5) and Streptococcus dysgalactiae (73.4%, 95% CI=60.9-83.7), with moderate Se for Streptococcus uberis (62.1%, 95% CI=49.3-73.8) and coagulase-negative staphylococci (59.8%, 95% CI=58.4-61.2). Sensitivities always increased as the number of affected quarters increased. Composite samples also showed high Sp (>97%) for most organisms. Factors such as lactation number and stage of lactation were evaluated for their influence on the Se and Sp of composite sampling, but were only found to be significant for coagulase-negative staphylococci. Predictive values using the herd prevalences found across Canada were calculated and can be useful in field scenarios when composite sampling is employed to assist mastitis management. When used to detect newly occurring IMI in pairs of samples taken before dry-off, post-calving, and also prior to and subsequent to the dry period, composite samples were shown to have lower Se but similar Sp for all pathogens investigated. Composite samples can be used to detect IMI and new IMI in dairy cows, but the Se and Sp of the procedure should be taken into account.

Reyher KK ，Dohoo IR 《-》

Diagnosing intramammary infections: comparison of multiple versus single quarter milk samples for the identification of intramammary infections in lactating dairy cows.

The objective was to examine the potential benefits of using different combinations of multiple quarter milk samples compared with a single sample for diagnosing intramammary infections (IMI) in dairy cattle. Data used in the analyses were derived from 7,076 samples from 667 quarters in 176 cows in 8 herds in 4 locations (Minnesota/Wisconsin, n=4; Prince Edward Island, n=2; Ontario, n=1; New York, n=1). Duplicate quarter milk samples were collected at morning milking for 5 consecutive days. Cows were evenly distributed between early postparturient and mid- to late-lactation cows. All samples were frozen for shipping and storage, thawed once, and cultured in university laboratories using standardized procedures consistent with National Mastitis Council guidelines. The presence of specific pathogens was confirmed and identified using the API identification system (bioMerieux, Marcy l'Etoile, France) in each laboratory. A previously developed gold standard was applied to the first sample from d 1, 3, and 5 to classify infected quarters. The data were analyzed separately for coagulase-negative staphylococci (CNS) and Streptococcus spp. Various combinations of test results from d 2 and 4 were used in the test evaluation. These consisted of single samples (n=4), 2 sets of duplicate samples (2 samples collected on the same day), 2 sets of consecutive samples (2 samples collected 2 d apart), and 2 sets of triplicate samples (2 samples on the same day and a third sample 2 d apart). Series interpretation of duplicate or consecutive samples (i.e., positive=same pathogen isolated from both samples) resulted in the highest specificity (Sp; CNS Sp=92.1-98.1%; Streptococcus spp. Sp=98.7-99.6%), but lowest sensitivity (Se; CNS Se=41.9-53.3%; Streptococcus spp. Se=7.7-22.2%). Parallel interpretation of duplicate or consecutive samples (i.e., positive=pathogen isolated from either) resulted in the highest Se (CNS Se=70.8-80.6%; Streptococcus spp. Se=31.6-48.1%), but lowest Sp (CNS Sp=72.0-77.3%; Streptococcus spp. Sp=89.5-93.3%). The difference in estimates between single and duplicate samples was larger than between single and consecutive samples. Overall, triplicate samples provided the best combination of Se and Sp, but compared with a single sample, provided only a modest gain in Sp and little or no gain in Se.

Dohoo I ，Andersen S ，Dingwell R ，Hand K ，Kelton D ，Leslie K ，Schukken Y ，Godden S ... -  《-》

Prevalence of mastitis pathogens in Ragusa, Sicily, from 2000 to 2006.

Ferguson JD ，Azzaro G ，Gambina M ，Licitra G ... -  《-》

Evaluation of minor pathogen intramammary infection, susceptibility parameters, and somatic cell counts on the development of new intramammary infections with major mastitis pathogens.

Major mastitis pathogens such as Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, and coliforms are usually considered more virulent and damaging to the udder than minor mastitis pathogens such as Corynebacterium spp. and coagulase-negative staphylococci (CNS). The current literature comprises several studies (n=38) detailing analyses with conflicting results as to whether intramammary infections (IMI) with the minor pathogens decrease, increase, or have no effect on the risk of a quarter acquiring a new IMI (NIMI) with a major pathogen. The Canadian Bovine Mastitis Research Network has a large mastitis database derived from a 2-yr data collection on a national cohort of dairy farms, and data from this initiative were used to further investigate the effect of IMI with minor pathogens on the acquisition of new major pathogen infections (defined as a culture-positive quarter sample in a quarter that had been free of that major pathogen in previous samples in the sampling period). Longitudinal milk samplings of clinically normal udders taken over several 6-wk periods as well as samples from cows pre-dry-off and postcalving were used to this end (n=80,397 quarter milk samples). The effects of CNS and Corynebacterium spp. on the major mastitis pathogens Staph. aureus, Strep. uberis, Strep. dysgalactiae, and coliform bacteria (Escherichia coli and Klebsiella spp.) were investigated using risk ratio analyses and multilevel logistic regression models. Quarter-, cow- and herd-level susceptibility parameters were also evaluated and were able to account for the increased susceptibility that exists within herds, cows and quarters, removing it from estimates for the effects of the minor pathogens. Increased quarter-level susceptibility was associated with increased risk of major pathogen NIMI for all pathogens except the coliforms. Increased somatic cell count was consistently associated with elevated risk of new major pathogen infections, but this was assumed to be a result of low sensitivity of bacteriology to diagnose major pathogen NIMI expediently and accurately. The presence of CNS in the sample 2 samplings before the occurrence of a NIMI increased the odds of experiencing a Staph. aureus NIMI 2.0 times, making the presence of CNS a risk factor for acquiring a Staph. aureus NIMI. Even with this extensive data set, power was insufficient to make a definitive statement about the effect of minor pathogen IMI on the acquisition of major pathogen NIMI. Definitively answering questions of this nature are likely to require an extremely large data set dedicated particularly to minor pathogen presence and NIMI with major pathogens.

Reyher KK ，Dohoo IR ，Scholl DT ，Keefe GP ... -  《-》

Sensitivity and specificity of PCR analysis and bacteriological culture of milk samples for identification of intramammary infections in dairy cows using latent class analysis.

Real-time PCR analysis of milk samples is a fast method to identify intramammary infections (IMI) in dairy cows, and has the potential to be used for routine analysis of test milking composite milk samples. However, the results of the PCR analysis can be difficult to interpret. The objective of this study was to compare the sensitivity (Se) and specificity (Sp) of PCR analysis of composite milk samples, and conventional bacteriological culturing (BC) of quarter milk samples, when used to identify cows with IMI. The comparisons were performed for IMI with four common udder pathogens; Staphylococcus aureus (S aureus), Streptococcus dysgalactiae (Str dysgalactiae), Str uberis and coagulase negative staphylococci (CoNS). The Se and Sp of real-time PCR (SePCR; SpPCR) and BC (SeBC; SpBC) was simultaneously estimated using latent class analysis (LCA), studying one pathogen at the time. Milk samples from 970 dairy cows from 25 herds were included. Aseptically collected quarter milk samples taken at the day before test milking (TM), at the day of TM, and at the day after TM, were analyzed using BC. Non-aseptically collected composite milk samples taken at the day of TM were analyzed using PCR. Moreover, the composite milk somatic cell count (SCC) was recorded and summarized by diagnostic test and bacterial finding. LCA was first performed using only test results from samples taken at the day of TM, but in a second analysis BC results from the three consecutive samplings, interpreted in parallel, were included. The SePCR was significantly higher than the SeBC for S aureus, Str dysgalactiae and CoNS in the first analysis, but only for CoNS in the second analysis. The SpPCR was significantly lower than the SpBC for Str dysgalactiae and CoNS. In conclusion, using PCR analysis of composite milk samples, as a diagnostic tool for identifying cows with IMI increased the Se for all the pathogens investigated (although not always significantly), while Sp in general remained on a similar level, compared to BC of quarter milk samples. The use of repeated quarter milk sampling improved the SeBC, making the results of PCR analysis and BC more similar. However, the SCC of cows with IMI according to BC was higher than for cows with IMI according to PCR, suggesting that some of the cows with IMI according to PCR did not have an active inflammation. Hence, extra caution is needed when decisions about treatment of IMI are based on PCR.

Nyman AK ，Persson Waller K ，Emanuelson U ，Frössling J ... -  《-》