Comparison of enzyme-linked immunosorbent assay and Fassisi® bovine immunoglobulin G (IgG) immunoassay for quantification of bovine IgG in neonatal calf serum.

BACKGROUND AND AIM: Rapid tests are routinely used to estimate serum immunoglobulin G (IgG) concentrations in diagnosing a failure of passive transfer (FPT) in calves. The study aimed to compare the Fassisi® Bovine IgG (FB-IgG) immunoassay and an enzyme-linked immunosorbent assay for quantifying bovine IgG in neonatal calf serum. MATERIALS AND METHODS: A total of 277 calves of 1-10 days of age were used in this study. Blood samples were obtained, and serum was extracted by centrifuging the samples at 2740× g for 5 min at 20°C. The serum was analyzed using the FB-IgG according to the manufacturer's specifications. Serum IgG concentrations were also determined by enzyme-linked immunosorbent assay (ELISA-IgG). FPT was defined as a serum IgG concentration <10 mg/mL. RESULTS: The mean ELISA-IgG serum concentration was 8.40 mg/mL (SD=7.02, range=0.10-47.50 mg/mL). FPT prevalence based on the ELISA measurements was 66.8%. The prevalence of partial and full FPT based on the FB-IgG was 54.5%. The ELISA-IgG and FB-IgG results were subjected to correlation and regression analysis. Overall sensitivity and specificity of the FB-IgG were 61.1% and 58.7%, respectively. A statistically significant dependence on age was identified in the results. CONCLUSION: Our findings suggest that the FB-IgG rapid method is less accurate and provides no other advantages over established methods.

[Influence factors on the results of a semiquantitative rapid test for immunoglobulin G concentration measurement in bovine calf serum]. / Beeinflussung des Ergebnisses eines semiquantitativen Schnelltests zur Ermittlung der Immunglobulin-G-Konzentration im Serum boviner Kälber.

OBJECTIVE: The Fassisi® Bovine IgG Test is a rapid blood test for the measurement of IgG levels in calves. The present study examined the influences of examiner subjectivity as well as time period between test begin and interpretation on the test results. MATERIALS AND METHODS: A single serum sample was obtained from each of 36 calves up to 10 days of age. Six veterinarians evaluated the serum samples obtained from 12 of the animals (inter-individual comparison). For the remaining 24 calves, the result was interpreted by one person after 10 minutes (manufacturer's information) as well as after 20 and 30 minutes (intra-individual comparison). RESULTS: In the inter-individual comparison, test interpretation differed in 4 out of 12 samples (33.3 %) between the different examiners. The intra-individual comparison showed that in 5 out of 24 samples (20.8 %) the test result were higher or lower when the time period from test begin to its interpretation was longer than the manufacturer's recommendation. In the majority of cases, the discrepancy amounted to maximally one level and the differences were not found to be statistically significant. CLINICAL RELEVANCE: Interpretation of the presented test was shown to be subject to subjective variations. Careful attention must be paid to adhering to the time period from test begin to its interpretation as specified by the manufacturer. Further studies verifying the presented results by a larger number of samples are warranted.

[Risk factors for calf mortality influence the occurrence of antibodies against the pathogens of enzootic bronchopneumonia]. / Risikofaktoren für die Kälbersterblichkeit beeinflussen den Nachweis von Antikörpern gegen die Erreger der enzootischen Bronchopneumonie.

OBJECTIVE: Bovine respiratory diseases are a common cause of calf loss. This study aimed to analyse associations between an occurrence of enzootic bronchopneumonia (EBP), calf mortality and calving management. MATERIAL AND METHODS: A total of 153 dairy farms participated in the study on a voluntary basis from November 2006 to July 2007. Calf management was inspected on-site during a farm visit and farm managers were required to complete a questionnaire on personal assessment of calving procedures, neonate management and environmental factors. Results were collated and matched with the calf mortality rate of 2006 determined from the HI-Tier database for each farm. Randomly selected serum samples of a mean number of 7 calves at the age 6 months per herd were investigated for antibodies against bovine respiratory syncytial virus (BRSV-AB) and parainfluenzavirus type 3 (PIV3-AB). According to the proportion of calves with BRSV-AB or PIV3-AB (≤ 20 % or > 20 %) farms were divided into 2 groups. RESULTS: Customary timing of the first colostrum feeding as well as the perceived level of importance of EBP to the farm manager, as described in the questionnaire, showed a positive correlation to calf mortality. BRSV-AB occurred more frequently on farms where managers stated that the first colostrum feeding occurred later than 4 hours after birth, that birth monitoring was rarely practiced and that the estimated level of dust in the calf barn was considered high. PIV3-AB was more frequently found at farms practicing tethered calving. CONCLUSION AND CLINICAL RELEVANCE: The results of this study indicate that peri- and postnatal calf management procedures may affect calf mortality and the frequency of occurrence of BRSV-AB or PIV3-AB. The influences of birth monitoring and the time of first colostrum feeding as well as dust exposure should be taken into account in future studies on the frequency of EBP and be included in the veterinary cause analysis of herd EBP-related problems.

The effect of skin fatty acids on Staphylococcus aureus.

Staphylococcus aureus is a commensal of the human nose and skin. Human skin fatty acids, in particular cis-6-hexadecenoic acid (C-6-H), have high antistaphylococcal activity and can inhibit virulence determinant production. Here, we show that sub-MIC levels of C-6-H result in induction of increased resistance. The mechanism(s) of C-6-H activity was investigated by combined transcriptome and proteome analyses. Proteome analysis demonstrated a pleiotropic effect of C-6-H on virulence determinant production. In response to C-6-H, transcriptomics revealed altered expression of over 500 genes, involved in many aspects of virulence and cellular physiology. The expression of toxins (hla, hlb, hlgBC) was reduced, whereas that of host defence evasion components (cap, sspAB, katA) was increased. In particular, members of the SaeRS regulon had highly reduced expression, and the use of specific mutants revealed that the effect on toxin production is likely mediated via SaeRS.

Structural analysis of Staphylococcus aureus serine/threonine kinase PknB.

Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 Å resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.

Surface display of Gaussia princeps luciferase allows sensitive fungal pathogen detection during cutaneous aspergillosis.

Non-invasive imaging techniques in microbial disease models have delivered valuable insights in the intimate pathogen-host interplay during infection. Here we describe evaluation and validation of a transgenic bioluminescence reporter strain of the human-pathogenic mold Aspergillus fumigatus, one of the main fungal pathogens affecting immunocompromised individuals. Expression and surface display of the Gaussia princeps luciferase allowed sensitive and rapid detection of luminescence emitted from this strain after substrate addition, with photon fluxes strongly correlating to the amounts of fungal conidia or germlings. The reporter strain allowed spatio-temporal monitoring of infection in a cutaneous model of aspergillosis, where neutropenic mice maintained the fungal burden while immunocompetent ones were able to clear it entirely. Most importantly, antifungal therapy could be followed in this type of disease model making use of the bioluminescent A. fumigatus strain. In conclusion, combining sensitivity of the Gaussia luciferase with a surface display expression system in the fungal host allows longitudinal infection studies on cutaneous forms of aspergillosis, providing perspective on drug screening approaches at high-throughput.

A metabolomic view of Staphylococcus aureus and its ser/thr kinase and phosphatase deletion mutants: involvement in cell wall biosynthesis.

Little is known about intracellular metabolite pools in pathogens such as Staphylococcus aureus. We have studied a particular metabolome by means of the presented LC-MS method. By investigating the central carbon metabolism which includes most of the energy transfer molecules like nucleotides, sugar mono- and biphosphates, and cofactors, a conclusion about phenotypes and stress answers in microorganisms is possible. Quantitative metabolite levels of S. aureus grown in complex lysogeny broth and in minimal medium were compared in the wild-type S. aureus strain 8325 and the isogenic eukaryotic-like protein serine/threonine kinase (DeltapknB) and phosphatase (Deltastp) deletion mutants. Detection of several remarkable differences, e.g., in nucleotide metabolism and especially cell wall precursor metabolites, indicates a previously unreported importance of serine/threonine kinase/phosphatase on peptidoglycan and wall teichoic acid biosynthesis. These findings may lead to new insights into the regulation of staphylococcal cell wall metabolism.

Staphylococcal PknB as the first prokaryotic representative of the proline-directed kinases.

In eukaryotic cell types, virtually all cellular processes are under control of proline-directed kinases and especially MAP kinases. Serine/threonine kinases in general were originally considered as a eukaryote-specific enzyme family. However, recent studies have revealed that orthologues of eukaryotic serine/threonine kinases exist in bacteria. Moreover, various pathogenic species, such as Yersinia and Mycobacterium, require serine/threonine kinases for successful invasion of human host cells. The substrates targeted by bacterial serine/threonine kinases have remained largely unknown. Here we report that the serine/threonine kinase PknB from the important pathogen Staphylococcus aureus is released into the external milieu, which opens up the possibility that PknB does not only phosphorylate bacterial proteins but also proteins of the human host. To identify possible human targets of purified PknB, we studied in vitro phosphorylation of peptide microarrays and detected 68 possible human targets for phosphorylation. These results show that PknB is a proline-directed kinase with MAP kinase-like enzymatic activity. As the potential cellular targets for PknB are involved in apoptosis, immune responses, transport, and metabolism, PknB secretion may help the bacterium to evade intracellular killing and facilitate its growth. In apparent agreement with this notion, phosphorylation of the host-cell response coordinating transcription factor ATF-2 by PknB was confirmed by mass spectrometry. Taken together, our results identify PknB as the first prokaryotic representative of the proline-directed kinase/MAP kinase family of enzymes.

Growth phase-dependent regulation of the global virulence regulator Rot in clinical isolates of Staphylococcus aureus.

Current models for global virulence regulation in Staphylococcus aureus are mainly based on studies performed with only a limited number of laboratory strains derived from NCTC8325. In these strains the small regulatory RNA, RNAIII, has a central role in virulence gene regulation. Recently, RNAIII was suggested to control transcription of target genes partly by inhibiting translation of the transcriptional regulator Rot. The present study was undertaken to examine if the model for RNAIII/Rot-dependent virulence regulation is conserved among clinical strains. To this end, we used Rot antibodies to directly assess the amount of Rot protein in 4 well-characterized S. aureus laboratory strains (8325-4, COL, Newman, and UAMS-1) and in 9 strains of clinical origin (encompassing USA300 and Mu50). Additionally, the cellular amount of RNAIII and rot mRNA was determined in all strains. The experiments revealed considerable variation in the Rot and RNAIII levels between strains. However, in the majority of strains the cellular amount of Rot was inversely correlated to the RNAIII level. As we demonstrate that Rot is a stable protein and that the level of rot transcript appeared similar in all strains, our data support that the model for RNAIII-mediated inhibition of rot mRNA translation is conserved among clinical strains. Assessment of Rot-dependent regulation of target genes revealed that Rot is a positive regulator of spa (protein A) transcription in all strains examined. In contrast, Rot repression of sspA (serine protease) and hlb (beta-hemolysin) transcription was not conserved between strains. From this study, we conclude that while the paradigm for understanding RNAIII-dependent regulation of Rot is well-conserved, regulation of single genes is subject to considerable strain variation. We propose that variation in global regulatory networks contribute considerably to the phenotypic variation observed between S. aureus isolates.

The impact of serine/threonine phosphorylation in Staphylococcus aureus.

In prokaryotes and eukaryotes, phosphotransfer represents a common mechanism to regulate cellular functions. Recent work revealed that modulation of cellular processes by eukaryote-like serine/threonine kinases (STKs) and phosphatases (STPs) are widespread in bacteria. During the last two years, first evidence on the role of Ser/Thr phosphorylation/dephosphorylation in Staphylococcus aureus has emerged leading to the identification of a functional STK and corresponding STP. Due to homology to known STKs/STPs in other bacterial species the kinase was designated PknB or alternatively Stk/Stk1, and the phosphatase Stp. The role of these enzymes in S. aureus has been examined by use of knock-out mutants and a kinase-overexpressing strain. These studies uncovered PknB/Stk and Stp as modulators of cell wall structure and susceptibility to cell wall-acting antibiotics such as certain beta-lactams and tunicamycin. By utilizing transcriptional profile analysis a strong regulatory impact of PknB/Stk on the expression of genes encoding proteins which are involved in purine and pyrimidine biosynthesis, cell wall metabolism, autolysis, and glutamine synthesis could be identified. Moreover, PknB/Stk is able to phosphorylate MgrA, thereby regulating activity of the efflux pump NorA. In a mouse pyelonephritis model PknB/Stk has been shown to play a role in virulence. Overall, Ser/Thr phosphorylation/dephosphorylation is a common theme in regulation of cellular functions determining metabolic activity and virulence also in the major human pathogen S. aureus.

Transcriptome and functional analysis of the eukaryotic-type serine/threonine kinase PknB in Staphylococcus aureus.

The function of the Staphylococcus aureus eukaryotic-like serine/threonine protein kinase PknB was investigated by performing transcriptome analysis using DNA microarray technology and biochemical assays. The transcriptional profile revealed a strong regulatory impact of PknB on the expression of genes encoding proteins which are involved in purine and pyrimidine biosynthesis, cell wall metabolism, autolysis, and glutamine synthesis. Functional activity of overexpressed and purified PknB kinase was demonstrated using the myelin basic protein as a surrogate substrate. Phosphorylation occurred in a time-dependent manner with Mn(2+) as a preferred cofactor. Furthermore, biochemical characterization revealed regulation of adenylosuccinate synthase (PurA) activity by phosphorylation. Phosphorylated PurA showed a 1.8-fold decrease in enzymatic activity compared to unphosphorylated PurA. Loss of PknB led to formation of larger cell clusters, and a pknB deletion strain showed 32-fold-higher sensitivity to the cell wall-active antibiotic tunicamycin. The results of this study strongly indicate that PknB has a role in regulation of purine biosynthesis, autolysis, and central metabolic processes in S. aureus.