Sensitivity and Specificity for the Detection of Clinical Mastitis by Automatic Milking Systems in Bavarian Dairy Herds

In automatic milking systems (AMSs), the detection of clinical mastitis (CM) and the subsequent separation of abnormal milk should be reliably performed by commercial AMSs. Therefore, the objectives of this cross-sectional study were (1) to determine the sensitivity (SN) and specificity (SP) of CM detection of AMS by the four most common manufacturers in Bavarian dairy farms, and (2) to identify routinely collected cow data (AMS and monthly test day data of the regional Dairy Herd Improvement Association (DHIA)) that could improve the SN and SP of clinical mastitis detection. Bavarian dairy farms with AMS from the manufacturers DeLaval, GEA Farm Technologies, Lely, and Lemmer-Fullwood were recruited with the aim of sampling at least 40 cows with clinical mastitis per AMS manufacturer in addition to clinically healthy ones. During a single farm visit, cow-level milking information was first electronically extracted from each AMS and then all lactating cows examined for their udder health status in the barn. Clinical mastitis was defined as at least the presence of visibly abnormal milk. In addition, available DHIA test results from the previous six months were collected. None of the manufacturers provided a definition for clinical mastitis (i.e., visually abnormal milk), therefore, the SN and SP of AMS warning lists for udder health were assessed for each manufacturer individually, based on the clinical evaluation results. Generalized linear mixed models (GLMMs) with herd as random effect were used to determine the potential influence of routinely recorded parameters on SN and SP. A total of 7411 cows on 114 farms were assessed; of these, 7096 cows could be matched to AMS data and were included in the analysis. The prevalence of clinical mastitis was 3.4% (239 cows). When considering the 95% confidence interval (95% CI), all but one manufacturer achieved the minimum SN limit of >80%: DeLaval (SN: 61.4% (95% CI: 49.0%–72.8%)), GEA (75.9% (62.4%–86.5%)), Lely (78.2% (67.4%–86.8%)), and Lemmer-Fullwood (67.6% (50.2%–82.0%)). However, none of the evaluated AMSs achieved the minimum SP limit of 99%: DeLaval (SP: 89.3% (95% CI: 87.7%–90.7%)), GEA (79.2% (77.1%–81.2%)), Lely (86.2% (84.6%–87.7%)), and Lemmer-Fullwood (92.2% (90.8%–93.5%)). All AMS manufacturers’ robots showed an association of SP with cow classification based on somatic cell count (SCC) measurement from the last two DHIA test results: cows that were above the threshold of 100,000 cells/mL for subclinical mastitis on both test days had lower chances of being classified as healthy by the AMS compared to cows that were below the threshold. In conclusion, the detection of clinical mastitis cases was satisfactory across AMS manufacturers. However, the low SP will lead to unnecessarily discarded milk and increased workload to assess potentially false-positive mastitis cases. Based on the results of our study, farmers must evaluate all available data (test day data, AMS data, and daily assessment of their cows in the barn) to make decisions about individual cows and to ultimately ensure animal welfare, food quality, and the economic viability of their farm

An E2-ubiquitin thioester-driven approach to identify substrates modified with ubiquitin and ubiquitin-like molecules.

Covalent modifications of proteins with ubiquitin and ubiquitin-like molecules are instrumental to many biological processes. However, identifying the E3 ligase responsible for these modifications remains a major bottleneck in ubiquitin research. Here, we present an E2-thioester-driven identification (E2~dID) method for the targeted identification of substrates of specific E2 and E3 enzyme pairs. E2~dID exploits the central position of E2-conjugating enzymes in the ubiquitination cascade and provides in vitro generated biotinylated E2~ubiquitin thioester conjugates as the sole source for ubiquitination in extracts. This enables purification and mass spectrometry-based identification of modified proteins under stringent conditions independently of the biological source of the extract. We demonstrate the sensitivity and specificity of E2-dID by identifying and validating substrates of APC/C in human cells. Finally, we perform E2~dID with SUMO in S. cerevisiae, showing that this approach can be easily adapted to other ubiquitin-like modifiers and experimental models

The Population Genetics of Pseudomonas aeruginosa Isolates from Different Patient Populations Exhibits High-Level Host Specificity

Objective To determine whether highly prevalent P. aeruginosa sequence types (ST) in Dutch cystic fibrosis (CF) patients are specifically linked to CF patients we investigated the population structure of P. aeruginosa from different clinical backgrounds. We first selected the optimal genotyping method by comparing pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and multilocus variable number tandem-repeat analysis (MLVA). Methods Selected P. aeruginosa isolates (n = 60) were genotyped with PFGE, MLST and MLVA to determine the diversity index (DI) and congruence (adjusted Rand and Wallace coefficients). Subsequently, isolates from patients admitted to two different ICUs (n = 205), from CF patients (n = 100) and from non-ICU, non-CF patients (n = 58, of which 19 were community acquired) were genotyped with MLVA to determine distribution of genotypes and genetic diversity. Results Congruence between the typing methods was >79% and DIs were similar and all >0.963. Based on costs, ease, speed and possibilities to compare results between labs an adapted MLVA scheme called MLVA9-Utrecht was selected as the preferred typing method. In 363 clinical isolates 252 different MLVA types (MTs) were identified, indicating a highly diverse population (DI = 0.995; CI = 0.993–0.997). DI levels were similarly high in the diverse clinical sources (all >0.981) and only eight genotypes were shared. MTs were highly specific (>80%) for the different patient populations, even for similar patient groups (ICU patients) in two distinct geographic regions, with only three of 142 ICU genotypes detected in both ICUs. The two major CF clones were unique to CF patients. Conclusion The population structure of P. aeruginosa isolates is highly diverse and population specific without evidence for a core lineage in which major CF, hospital or community clones co-cluster. The two genotypes highly prevalent among Dutch CF patients appeared unique to CF patients, suggesting specific adaptation of these clones to the CF lung

Review of the Palaearctic species of Ismaridae Thomson, 1858 (Hymenoptera: Diaprioidea)

This is an open access article, available to all readers online, published under a Creative Commons BY-NC-ND license: https://creativecommons.org/licenses/by-nc-nd/3.0/. The attached file is the published version of the article

Fatal Pseudomonas aeruginosa pneumonia in a previously healthy woman was most likely associated with a contaminated hot tub

Community-acquired pneumonia due to Pseudomonas aeruginosa in previously healthy individuals is a rare disease that is associated with high fatality. On 14 February 2010 a previously healthy 49-year-old woman presented to an emergency room with signs and symptoms of pneumonia, 2 days after returning from a spa holiday in a wellness hotel. Blood cultures and respiratory specimens grew P. aeruginosa. Despite adequate antimicrobial therapy, the patient died of septic multiorgan failure on day nine of hospitalization. On February 26, nine water samples were taken from the hotel facilities used by the patient: In the hot tub sample 37,000 colony-forming units of P. aeruginosa/100 ml were detected. Two of five individual colonies from the primary plate used for this hot tub water sample were found to be genetically closely related to the patients’ isolates. Results from PFGE, AFLP and MLST analysis allowed the two lung isolates gained at autopsy and the whirlpool bathtub isolates to be allocated into one cluster. The patient most likely acquired P. aeruginosa from the contaminated water in the hotel’s hot tub. The detection of P. aeruginosa in high numbers in a hot tub indicates massive biofilm formation in the bath circulation and severe deficiencies in hygienic maintenance. The increasing popularity of hot tubs in hotels and private homes demands increased awareness about potential health risks associated with deficient hygienic maintenance

The evolution of bicontinuous polymeric nanospheres in aqueous solution

Complex polymeric nanospheres in aqueous solution are desirable for their promising potential in encapsulation and templating applications. Understanding how they evolve in solution enables better control of the final structures. By unifying insights from cryoTEM and small angle X-ray scattering (SAXS), we present a mechanism for the development of bicontinuous polymeric nanospheres (BPNs) in aqueous solution from a semi-crystalline comb-like block copolymer that possesses temperature-responsive functionality. During the initial stages of water addition to THF solutions of the copolymer the aggregates are predominantly vesicles; but above a water content of 53% irregular aggregates of phase separated material appear, often microns in diameter and of indeterminate shape. We also observe a cononsolvency regime for the copolymer in THF–water mixtures from 22 to 36%. The structured large aggregates gradually decrease in size throughout dialysis, and the BPNs only appear upon cooling the fully aqueous dispersions from 35 °C to 5 °C. Thus, the final BPNs are ultimately the result of a reversible temperature-induced morphological transition

TRIP13 and APC15 drive mitotic exit by turnover of interphase- and unattached kinetochore-produced MCC

The mitotic checkpoint ensures accurate chromosome segregation through assembly of the mitotic checkpoint complex (MCC), a soluble inhibitor of the anaphase-promoting complex/cyclosome (APC/C) produced by unattached kinetochores. MCC is also assembled during interphase by Mad1/Mad2 bound at nuclear pores, thereby preventing premature mitotic exit prior to kinetochore maturation and checkpoint activation. Using degron tagging to rapidly deplete the AAA+ ATPase TRIP13, we show that its catalytic activity is required to maintain a pool of open-state Mad2 for MCC assembly, thereby supporting mitotic checkpoint activation, but is also required for timely mitotic exit through catalytic disassembly of MCC. Strikingly, combining TRIP13 depletion with elimination of APC15-dependent Cdc20 ubiquitination/degradation results in a complete inability to exit mitosis, even when MCC assembly at unattached kinetochores is prevented. Thus, mitotic exit requires MCC produced either in interphase or mitosis to be disassembled by TRIP13-catalyzed removal of Mad2 or APC15-driven ubiquitination/degradation of its Cdc20 subunit

APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment

Faithful chromosome segregation during mitosis depends on the Spindle Assembly Checkpoint (SAC) that monitors kinetochore attachment to the mitotic spindle. Unattached kinetochores generate mitotic checkpoint proteins complexes (MCCs) that bind and inhibit the Anaphase Promoting Complex/Cyclosome (APC/C). How the SAC proficiently inhibits the APC/C but still allows its rapid activation when the last kinetochore attaches to the spindle is important to understand how cells maintain genomic stability. We show that the APC/C subunit APC15 is required for the turnover of the APC/C co-activator Cdc20 and release of MCCs during SAC signalling but not for APC/C activity per se. In the absence of APC15, MCCs and ubiquitylated Cdc20 remain ‘locked’ onto the APC/C, which prevents the ubiquitylation and degradation of Cyclin B1 when the SAC is satisfied. We conclude that APC15 mediates the constant turnover of Cdc20 and MCCs on the APC/C to allow the SAC to respond to the attachment state of kinetochores

Conditional targeting of MAD1 to kinetochores is sufficient to reactivate the spindle assembly checkpoint in metaphase

Fidelity of chromosome segregation is monitored by the spindle assembly checkpoint (SAC). Key components of the SAC include MAD1, MAD2, BUB1, BUB3, BUBR1, and MPS1. These proteins accumulate on kinetochores in early prometaphase but are displaced when chromosomes attach to microtubules and/or biorient on the mitotic spindle. As a result, stable attachment of the final chromosome satisfies the SAC, permitting activation of the anaphase promoting complex/cyclosome (APC/C) and subsequent anaphase onset. SAC satisfaction is reversible, however, as addition of taxol during metaphase stops cyclin B1 degradation by the APC/C. We now show that targeting MAD1 to kinetochores during metaphase is sufficient to reestablish SAC activity after initial silencing. Using rapamycin-induced heterodimerization of FKBP-MAD1 to FRB-MIS12 and live monitoring of cyclin B1 degradation, we show that timed relocalization of MAD1 during metaphase can stop cyclin B1 degradation without affecting chromosome-spindle attachments. APC/C inhibition represented true SAC reactivation, as FKBP-MAD1 required an intact MAD2-interaction motif and MPS1 activity to accomplish this. Our data show that MAD1 kinetochore localization dictates SAC activity and imply that SAC regulatory mechanisms downstream of MAD1 remain functional in metaphase. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00412-014-0458-9) contains supplementary material, which is available to authorized users