Evaluation of the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) system in the detection of mastitis pathogens from bovine milk samples.

MALDI-TOF is a chemistry analytical tool that has recently been deployed in the identification of microorganisms isolated from nosocomial environments. Its use in diagnostics has been extremely advantageous in terms of cost effectiveness, sample preparation easiness, turn-around time and result analysis accessibility. In the dairy industry, where mastitis causes great financial losses, a rapid diagnostic method such as MALDI-TOF could assist in the control and prevention program of mastitis, in addition to the sanitation and safety level of the dairy farms and processing facility. However, the diagnostic strengths and limitations of this test method require further understanding. In the present study, we prospectively compared MALDI-TOF MS to conventional 16S rDNA sequencing method for the identification of pathogens recovered from milk associated with clinical and subclinical bovine mastitis cases. Initially, 810 bacterial isolates were collected from raw milk samples over a period of three months. However, only the isolates (481) having both 16S rDNA sequencing and MALDI-TOF identification were included in the final phase of the study. Among the 481 milk isolates, a total of 26 genera (12 g-postive and 14 g-negative), including 71 different species, were taxonomically charecterized by 16S rDNA at the species level. Comparatively, MALDI-TOF identified 17 genera (9 g-positive and 8 g-negative) and 33 differernt species. Overall, 445 (93%) were putatively identified to the genus level by MALDI-TOF MS and 355 (74%) were identified to the species level, but no reliable identification was obtained for 16 (3.3%), and 20 (4.2%) discordant results were identified. Future studies may help to overcome the limitations of the MALDI database and additional sample preparation steps might help to reduce the number of discordances in identification. In conclusion, our results show that MALDI-TOF MS is a fast and reliable technique which has the potential to replace conventional identification methods for common mastitis pathogens, routinely isolated from raw milk. Thus it's adoption will strengthen the capacity, quality, and possibly the scope of diagnostic services to support the dairy industry.

Evaluation of rapid culture, a predictive algorithm, esterase somatic cell count and lactate dehydrogenase to detect intramammary infection in quarters of dairy cows at dry-off.

Our objective was to compare four tests to standard milk culture followed by MALDI-ToF in quarters of cows at dry-off. Cows (n = 432) were randomly selected from seven U.S. dairy herds already participating in a multi-site clinical trial in summer 2018. Aseptic foremilk samples were collected from quarters (n = 1728) two days prior to dry-off, and subjected to index and reference tests. The four index tests included rapid culture, a predictive algorithm, an esterase strip test measuring somatic cell count (SCC) and a cow-side lactate dehydrogenase (LDH) test. Rapid culture was performed by inoculating quarter milk samples onto a commercial rapid culture plate. Plates were evaluated by technicians after 30-40 h of incubation at 37 ± 2 °C. Quarters were classified as infected if any bacterial growth was observed. For the algorithm test method, all quarters were classified as infected if the cow met any of the following criteria: 1) any Dairy Herd Improvement Association (DHIA) test with a SCC > 200,000 cells / ml during the current lactation or 2) two or more clinical mastitis cases during the current lactation. Esterase-SCC and cow-side LDH tests involved adding milk to the test strip and reading for color changes. For esterase-SCC and cow-side LDH tests, low (≥250 cells / ml and ≥100 U / L) and high (≥500 cells / ml and ≥200 U / L) thresholds were used to classify quarters as infected or not. Composite samples (4 × 2 mL quarter-milk samples commingled) were also tested for rapid culture, esterase-SCC and cow-side LDH tests, such that if a composite sample was positive, then all quarters contributing to that sample were classified as infected. The reference test was traditional aerobic culture conducted in an accredited laboratory using MALDI-ToF for identification of isolates. Traditional culture was only conducted on quarter-milk samples, and consequently, IMI was always considered at the quarter-level. Unconditional logistic regression was used to estimate sensitivity (SE), specificity (SP), apparent prevalence, positive predictive values (PPV) and negative predictive values (NPV) for each index test. Cohen's Kappa (κ) was used to measure agreement between tests. Algorithm, esterase-SCC and cow-side LDH tests had poor agreement with the reference test (κ ranging from 0.01 to 0.12), while rapid culture had fair agreement (κ = 0.28). No test had concurrently high SE and SP. Negative predictive values were moderate to high for all tests.

The impact of federal bioterrorism funding programs on local health department preparedness activities.

Using the 2005 National Association of County and City Health Officers Profile of Local Health Departments data set, bivariate probit and Heckman selection models were used to test the hypothesis that the level of federal funding received for bioterrorism preparedness is related to the preparedness activities undertaken by local health departments. Overall budget, leadership, and crisis experience are found to be the most important determinants of local preparedness activity, but Centers for Disease Control and Prevention preparedness funding plays a mediating role by building capacity through the hiring of one key leadership position, the emergency preparedness coordinator. Additional research is needed to determine the potential impact of these funds on other aspects of the local public health system, such as the scope of services delivered, to determine secondary effects of the program.

C-reactive protein does not relax vascular smooth muscle: effects mediated by sodium azide in commercially available preparations.

C-reactive protein (CRP), an acute-phase protein and newly recognized indicator of cardiovascular risk, may have direct actions on the vascular wall. Previous studies suggest that CRP is a vasodilator that activates smooth muscle K(+) channels. We examined the reported vasoactive properties of CRP and further explored its mechanisms of action. CRP decreased blood pressure in rats and increased coronary flow in open-chest dogs at a constant coronary perfusion pressure. CRP relaxed rat aortic rings and mesenteric small arteries that were contracted with phenylephrine. Relaxation was not affected by endothelial denudation or inhibition of nitric oxide (NO) synthase but was blocked by inhibition of soluble guanylate cyclase or K(+) channels. CRP solutions remained effective, i.e., elicited vasodilation, even after boiling or enzymatic digestion, which suggests the presence of a nonprotein contaminant. Sodium azide (NaN(3), 0.1%) is the preservative used for commercially available CRP and a potential source of NO. NaN(3) elicited the same cardiovascular effects as CRP preparations at equal concentrations, and its actions were blocked by inhibition of guanylate cyclase and K(+) channels. NaN(3)-free CRP, prepared by gel-filtration centrifugation and confirmed by electrophoresis, had no effect on vascular tone. Inhibition of vascular smooth muscle catalase with 3-amino-1,2,4-triazole completely prevented the effects of NaN(3) and NaN(3)-containing CRP solutions. We demonstrate that the acute vasoactive properties of commercially available CRP preparations are attributable to NaN(3) (and subsequent production of NO by catalase); therefore, this study suggests a reappraisal of the acute role of CRP in regulating vascular tone.