Performance evaluation of a rapid immunochromatographic test kit in detecting bovine mastitis-causing streptococci.

Mastitis causes significant economic losses to the dairy industry due to decreased milk production in infected cows. Identification of mastitis-causing pathogens, such as streptococci, is necessary for selecting an effective antibiotic for treating mastitis. Although bacterial cultivation is widely used for pathogen identification, it requires more than 24 hr to complete. Contrarily, Lateral flow assays are simple, rapid, and inexpensive testing procedures. In this study, the effectiveness of an immunochromatographic test kit for detecting streptococci in milk samples from cows with clinical mastitis was evaluated as an alternative to bacterial cultivation. The performance of the immunochromatographic test kit for detecting mastitis-causing pathogens was compared with that of bacterial cultivation and real-time quantitative polymerase chain reaction (qPCR). The sensitivity and specificity of the immunochromatographic test kit were 0.800 and 0.875, respectively, compared with bacterial cultivation. Additionally, the κ statistic values of the immunochromatographic test kit was 0.667, indicating substantial agreement with the results of bacterial cultivation. Statistically, sensitivity and specificity of the immunochromatographic kit and real-time qPCR did not differ significantly; thus, the immunochromatographic test kit detected mastitis-causing streptococci as effectively as real-time qPCR. Therefore, the immunochromatographic kit is a rapid, inexpensive, and simple method for detecting streptococci and contributes to the timely selection of appropriate antibiotics for treatment and promotes early recovery from mastitis.

A novel vaccine strategy using quick and easy conversion of bacterial pathogens to unnatural amino acid-auxotrophic suicide derivatives.

We propose a novel strategy for quick and easy preparation of suicide live vaccine candidates against bacterial pathogens. This method requires only the transformation of one or more plasmids carrying genes encoding for two types of biological devices, an unnatural amino acid (uAA) incorporation system and toxin-antitoxin systems in which translation of the antitoxins requires the uAA incorporation. Escherichia coli BL21-AI laboratory strains carrying the plasmids were viable in the presence of the uAA, whereas the free toxins killed these strains after the removal of the uAA. The survival time after uAA removal could be controlled by the choice of the uAA incorporation system and toxin-antitoxin systems. Multilayered toxin-antitoxin systems suppressed escape frequency to less than 1 escape per 109 generations in the best case. This conditional suicide system also worked in Salmonella enterica and E. coli clinical isolates. The S. enterica vaccine strains were attenuated with a >105 fold lethal dose. Serum IgG response and protection against the parental pathogenic strain were confirmed. In addition, the live E. coli vaccine strain was significantly more immunogenic and provided greater protection than a formalin-inactivated vaccine. The live E. coli vaccine was not detected after inoculation, presumably because the uAA is not present in the host animals or the natural environment. These results suggest that this strategy provides a novel way to rapidly produce safe and highly immunogenic live bacterial vaccine candidates. IMPORTANCE: Live vaccines are the oldest vaccines with a history of more than 200 years. Due to their strong immunogenicity, live vaccines are still an important category of vaccines today. However, the development of live vaccines has been challenging due to the difficulties in achieving a balance between safety and immunogenicity. In recent decades, the frequent emergence of various new and old pathogens at risk of causing pandemics has highlighted the need for rapid vaccine development processes. We have pioneered the use of uAAs to control gene expression and to conditionally kill host bacteria as a biological containment system. This report proposes a quick and easy conversion of bacterial pathogens into live vaccine candidates using this containment system. The balance between safety and immunogenicity can be modulated by the selection of the genetic devices used. Moreover, the uAA-auxotrophy can prevent the vaccine from infecting other individuals or establishing the environment.

Association of resting energy expenditure with phase angle in hospitalized older patients: a cross-sectional analysis.

BACKGROUND/OBJECTIVES: Resting energy expenditure (REE) constitutes the largest component of total energy expenditure and undergoes an age-related decline that is unexplained by decreased fat-free mass. Phase angle (PhA) is a cellular health indicator that is possibly associated with REE. We investigated the association of REE and PhA in hospitalized older adults. SUBJECTS/METHODS: This single-center, cross-sectional analysis utilized the baseline data from a prospective longitudinal study and included 131 eligible patients aged ≥70 years. The REE was measured using indirect calorimetry, and PhA and body composition were assessed using bioelectrical impedance. The association between REE, PhA, and body composition was examined, and REE was compared using previously reported PhA cutoff values. RESULTS: In this cohort with a mean (±standard deviation) age of 87.4 (±7.0) years, 34.4% of the participants were men. REE and PhA correlated strongly (r: 0.562, p < 0.001) and significantly after adjusting for age and sex (r: 0.433, p < 0.001). Multivariate analysis showed a significant independent association between REE and PhA and skeletal muscle mass (standardized ß [95% CI]; 28.072 [2.188-53.956], p = 0.035) without any significant interaction between PhA and age on REE. The low PhA group had a significantly lower REE (kcal/day; 890 [856-925] vs. 1077 [1033-1122], p < 0.001), and this remained significant after adjusting for age, sex, and skeletal muscle mass index. CONCLUSIONS: PhA is associated with REE in older adults. Adjusting REE calculation algorithms based on PhA values and correcting predicted REE according to PhA may aid in determining more accurate energy requirements.

New prediction equations for resting energy expenditure in older hospitalized patients: Development and validation.

OBJECTIVES: Accurate resting energy expenditure (REE) prediction is needed to prevent over- or underfeeding in older hospitalized patients. However, few validated REE prediction Equations are known for such patients. Therefore, this study aimed to develop new REE prediction Equations and evaluate their validity. METHODS: This single-center, cross-sectional study enrolled 134 patients ages ≥70 y. For holdout validation, patients were randomized in a 3:1 ratio; for the development data set, a new Equation was developed according to the measured REE using indirect calorimetry. The new and existing Equations were compared using the validation data set. RESULTS: Mean patient age was 87.4 ± 6.9 y, and 34.3% were male. Two Equations were developed in multivariable regression models: Equation 1: REE (kcal/day) = 313.582 + Height (cm) × 3.973 + Body weight (kg) × 5.332 - Age (y) × 5.474 - (0 if male; 1 if female) × 20.012 + Calf circumference (cm) × 12.174; and Equation 2: REE (kcal/day) = 594.819 + Height (cm) × 3.760 + Body weight (kg) × 8.888 - Age (y) × 6.298 - (0 if male; 1 if female) × 16.396. The mean relative bias (95% CI) with measured REE as a reference had a small bias for Equations 1 and 2 (-0.1 [-4.1 to 3.9]% and -0.2 [-4.4 to 4.1]%, respectively); however, the Harris-Benedict, Food and Agriculture Organization of the United Nations/World Health Organization/United Nations University, Ganpule, and body weight × 20 Equations had larger biases (-6.2 [-10.3 to -2.0]%; 5.3 [1.3 to 9.3]%; -13.9 [-18.6 to -9.3]%; and -11.6 [-16.1 to -7.1]%, respectively). CONCLUSIONS: New prediction Equations using height, body weight, age, sex, and calf circumference improve REE prediction accuracy in older hospitalized patients.

Resting Energy Expenditure in Older Inpatients: A Comparison of Prediction Equations and Measurements.

Determining energy requirements are an important component of nutritional support for patients with malnutrition; however, the validity of prediction equations for resting energy expenditure (REE) is disputed in older hospitalized patients. We aimed to assess the validity of these equations in older hospitalized patients in Japan. This was a single-center, cross-sectional study of 100 patients aged ≥70 years, hospitalized between January 2020 and December 2021. REE was measured using an indirect calorimeter and was compared to the predicted values calculated from five REE prediction equations. The mean (95% confidence interval) measured REE was 968.1 (931.0, 1005.3) kcal/day, and the mean predicted REE was higher for the FAO/WHO/UNU (1014.3 [987.1, 1041.6] kcal/day, p = 0.164) and Schofield (1066.0 [1045.8, 1086.2] kcal/day, p < 0.001) equations and lower for the Harris-Benedict (898.6 [873.1, 924.1] kcal/day, p = 0.011), Ganpule (830.1 [790.3, 869.9] kcal/day, p < 0.001), and body weight (kg) × 20 (857.7 [821.9, 893.5] kcal/day, p < 0.001) equations. In the age group analysis, none of the predicted values were within a 10% error for more than 80% of patients aged 70−89 years and ≥90 years. The five REE prediction equations did not provide accurate estimates. Validated REE prediction equations need to be developed for older hospitalized patients.

Structural comparison of the C-terminal domain of functionally divergent lyssavirus P proteins.

Lyssavirus P protein is a multifunctional protein that interacts with numerous host-cell proteins. The C-terminal domain (CTD) of P is important for inhibition of JAK-STAT signaling enabling the virus to evade host immunity. Several regions on the surface of rabies virus P are reported to interact with host factors. Among them, an extended, discrete hydrophobic patch of P CTD is notable. Although structures of P CTD of two strains of rabies virus, and of mokola virus have been solved, the structure of P CTD for Duvenhage virus, which is functionally divergent from these species for immune evasion function, is not known. Here, we analyze the structures of P CTD of Duvenhage and of a distinct rabies virus strain to gain further insight on the nature and potential function of the hydrophobic surface. Molecular contacts in crystals suggest that the hydrophobic patch is important to intermolecular interactions with other proteins, which differ between the lyssavirus species.

The Measles Virus V Protein Binding Site to STAT2 Overlaps That of IRF9.

Measles virus (MeV) is a highly immunotropic and contagious pathogen that can even diminish preexisting antibodies and remains a major cause of childhood morbidity and mortality worldwide despite the availability of effective vaccines. MeV is one of the most extensively studied viruses with respect to the mechanisms of JAK-STAT antagonism. Of the three proteins translated from the MeV P gene, P and V are essential for inactivation of this pathway. However, the lack of data from direct analyses of the underlying interactions means that the detailed molecular mechanism of antagonism remains unresolved. Here, we prepared recombinant MeV V protein, which is responsible for human JAK-STAT antagonism, and a panel of variants, enabling the biophysical characterization of V protein, including direct V/STAT1 and V/STAT2 interaction assays. Unambiguous direct interactions between the host and viral factors, in the absence of other factors such as Jak1 or Tyk2, were observed, and the dissociation constants were quantified for the first time. Our data indicate that interactions between the C-terminal region of V and STAT2 is 1 order of magnitude stronger than that of the N-terminal region of V and STAT1. We also clarified that these interactions are completely independent of each other. Moreover, results of size exclusion chromatography demonstrated that addition of MeV-V displaces STAT2-core, a rigid region of STAT2 lacking the N- and C-terminal domains, from preformed complexes of STAT2-core/IRF-associated domain (IRF9). These results provide a novel model whereby MeV-V can not only inhibit the STAT2/IRF9 interaction but also disrupt preassembled interferon-stimulated gene factor 3.IMPORTANCE To evade host immunity, many pathogenic viruses inactivate host Janus kinase signal transducer and activator of transcription (STAT) signaling pathways using diverse strategies. Measles virus utilizes P and V proteins to counteract this signaling pathway. Data derived largely from cell-based assays have indicated several amino acid residues of P and V proteins as important. However, biophysical properties of V protein or its direct interaction with STAT molecules using purified proteins have not been studied. We have developed novel molecular tools enabling us to identify a novel molecular mechanism for immune evasion whereby V protein disrupts critical immune complexes, providing a clear strategy by which measles virus can suppress interferon-mediated antiviral gene expression.