Serum bactericidal titres for monitoring antimicrobial therapy: current status and potential role in the management of multidrug-resistant Gram-negative infections.

BACKGROUND: Serum bactericidal titres (SBTs) were widely used in the 1970s and 1980s to monitor antimicrobial therapy but are now seldom recommended. It is the only laboratory test that integrates drug pharmacodynamics, host pharmacokinetics and synergistic or antagonistic interactions of antimicrobial combinations into a single index of antimicrobial activity. We hypothesized that SBTs could play a renewed role in monitoring antibiotic treatment of multidrug-resistant Gram-negative infections. However, the last critical appraisal of the test was published over 30 years ago. OBJECTIVES: This narrative review provides an updated assessment of the SBT test and its methodological limitations. We performed a diagnostic meta-analysis to estimate the value of SBTs for predicting clinical failure or death during antibiotic treatment. SOURCES: A comprehensive literature search of PubMed including all English publications was performed in December 2019 using the Medical Subject Headings (MeSH search terms "serum", "bactericidal", "inhibitory", "titre", "monitoring", "anti-infective agents" "antimicrobial therapy" and "therapeutic drug monitoring"). CONTENT: Although standardized methods for performing SBTs were approved in 1999, the test remains labour intensive, and results may not be available until 72 hr. However, the use of non-culture-based endpoints (i.e. spectrophotometric or fluorescent) may shorten test time to 24 hr. Despite considerable heterogeneity in published studies, a meta-analysis of 11 evaluable studies published from 1974 to 2007 indicated a critical SBT result (peak SBT ≤1:8 or trough ≤1:2) is associated with a diagnostic odds ratio for clinical failure during antibiotic treatment of 12.27 (95% confidence interval 5.28-28.54) and a 5.32 (95% 1.32-21.42) odds of death. IMPLICATIONS: SBTs have prognostic value for identifying patients at high risk for antibiotic treatment failure, but the slow turnaround time of the current test limits its clinical utility. Standardization of a more rapid SBT testing method is needed.

Immune status of Oreochromis niloticus subjected to long-term lead nitrate exposure and a Arthrospira platensis treatment trial.

In this study, the impacts of lead toxicity on Oreochromisniloticus were investigated. Additionally, the potential ameliorative effects of the Spirulina algae Arthrospira platensis were evaluated. The median lethal concentration (LC50) of PbNO3 was determined to be 143.3 mg/l for O. niloticus weighing 42 ± 2.5 g. O. niloticus were exposed to 10 % of the estimated PbNO3 LC50 for 12 weeks. The cumulative mortality rate (CMR) increased with exposure time. The results of assays for red blood cells (RBCs), haemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC) indicated that the exposed O. niloticus suffered from anaemia. The levels of liver enzymes, namely, aspartate transaminase (AST) and alanine transaminase (ALT), as well as metallothionein)MT(revealed deterioration of hepatic tissue. The activity of the antioxidant enzymes glutathione peroxidase (GPx) as well as catalase (CAT) was stimulated in the hepatic tissue of O. niloticus exposed to PbNO3 and in those treated with A. platensis. Based on the results of serum bactericidal activity (SBA) and oxidative burst activity (OBA) assays as well as challenge tests with Aeromonas hydrophila, it was clear that supplementation with 5 or 10 g/kg A. platensis significantly enhanced the fish immune status and decreased the mortality rate (MR). However, these effects were reduced by PbNO3 exposure with no differences in MR percentage. Therefore, it was clear that O. niloticus reared in lead nitrate-polluted water were immunosuppressed, while diet supplementation with A. platensis could ameliorate such impacts.

Altitudinal Effects on Innate Immune Response of a Subterranean Rodent.

Immune defense is costly to maintain and deploy, and the optimal investment into immune defense depends on risk of infection. Altitude is a natural environmental factor that is predicted to affect parasite abundance, with lower parasite abundance predicted at higher altitudes due to stronger environmental stressors, which reduce parasite transmission. Using high and low altitude populations of the Turkish blind mole-rat (TBMR) Nannospalax xanthodon, we tested for effects of altitude on constitutive innate immune defense. Field studies were performed with 32 wild animals in 2017 and 2018 from two low- and one high-altitude localities in the Central Taurus Mountains, at respective altitudes of 1010 m, 1115 m, and 2900 m above sea level. We first compared innate standing immune defense as measured by the bacteria-killing ability of blood serum. We then measured corticosterone stress hormone levels, as stressful conditions may affect immune response. Finally, we compared prevalence and intensity of gastrointestinal parasites of field-captured TBMR. We found that the bacteria-killing ability of serum is greater in the mole-rat samples from high altitude. There was no significant difference in stress (corticosterone) levels between altitude categories. Coccidian prevalence and abundance were significantly higher in 2017 than 2018 samples, but there was no significant difference in prevalence, abundance, or intensity between altitudes, or between sexes. Small sample sizes may have reduced power to detect true differences; nevertheless, this study provides support that greater standing innate immunity in high altitude animals may reflect greater investment into constitutive defense.

Is it possible to shorten serum bactericidal testing?

Serum bactericidal test represents an alternative possibility for optimization of antibiotic treatment. The paper aimed to confirm non-inferiority of bactericidal testing using the broth dilution method according to the CLSI method (M21A) in comparison with turbidimetric and colorimetric modifications. We tested human blood sera (n = 76) of ten hematological patients, their blood was withdrawn prior to and during the course of antibiotic therapy. Testing employed the reference strain Escherichia coli ATCC 25922. The results of the modified turbidimetric method did not differ in a statistically significant way with the use of the wavelengths of 620 nm or 405 nm and the break-point <30% turbidity change after 24-hour incubation. The colorimetric method was also non-inferior from the CLSI method when resazurin was applied after 8-hour incubation and the results of subculture were read after 24-hour incubation. Both tested modifications can represent a shorter alternative to the CLSI reference method.

Exposure to fluctuating temperatures leads to reduced immunity and to stress response in rattlesnakes.

Ectothermic organisms often experience considerable variation in their body temperature throughout the circadian cycle. However, studies focusing on the measurement of physiological traits are usually performed under constant temperature regimes. This mismatch between thermal exposure in the field and experimental conditions could act as a stressor agent, as physiological functions are strongly influenced by temperature. Herein, we asked the question whether constant thermal regimes would cause a stress response and impact the immunity of the South American rattlesnake (Crotalus durissus) when compared with a fluctuating thermal regime. We addressed this question by determining heterophil:lymphocyte (H:L) ratio, plasma bacteria-killing ability (BKA) and corticosterone (CORT) levels in snakes kept under a constant temperature regime at 30°C, and under a fluctuating regime that oscillated between 25°C at night and 35°C during the day. The experiments had a mirrored design, in which half of the snakes were subjected to a fluctuating-to-constant treatment, while the other half was exposed to a constant-to-fluctuating treatment. The shift from constant to fluctuating thermal regime was accompanied by an increase in plasma CORT levels, indicating the activation of a stress response. Exposure to a fluctuating thermal regime at the onset of the experiments induced a decrease in the BKA of rattlesnakes. H:L ratio was not affected by treatments and, therefore, the shift between thermal regimes seems to have acted as a low-intensity stressor. Our results suggest that removal from temperatures close to the snake's preferred body temperature triggers a stress response in rattlesnakes.

Chimeric hepatitis B virus core particles displaying Neisserial surface protein A confer protection against virulent Neisseria meningitidis serogroup B in BALB/c mice.

PURPOSE: The primary goal of the present study was to explore and evaluate the highly conserved Neisserial surface protein A (NspA) molecule, fused with truncated HBV virus-like particles (VLPs), as a candidate vaccine against the virulent Neisseria meningitidis serogroup B (NMB). METHODS: NspA was inserted into the major immunodominant region of the truncated hepatitis B virus core protein (HBc; amino acids 1-144). The chimeric protein, HBc-N144-NspA, was expressed from a prokaryotic vector and generated HBc-like particles, as determined by transmission electron microscopy. Further, the chimeric protein and control proteins were used to immunize mice and the resulting immune responses evaluated by flow cytometry, enzyme-linked immunosorbent assay, and analysis of serum bactericidal activity (SBA) titer. RESULTS: Evaluation of the immunogenicity of the recombinant HBc-N144-NspA protein showed that it elicited the production of high levels of NspA-specific total IgG. The SBA titer of HBc-N144-NspA/F reached 1:16 2 weeks after the last immunization in BALB/c mice, when human serum complement was included in the vaccine. Immunization of HBc-N144-NspA, even without adjuvant, induced high levels of IL-4 and a high IgG1 to IgG2a ratio, confirming induction of an intense Th2 immune response. Levels of IL-17A increased rapidly in mice after the first immunization with HBc-N144-NspA, indicating the potential for this vaccine to induce a mucosal immune response. Meanwhile, the immunization of HBc-N144-NspA without adjuvant induced only mild inflammatory infiltration into the mouse muscle tissue. CONCLUSION: This study demonstrates that modification using HBc renders NspA a candidate vaccine, which can trigger protective immunity against NMB.

The bivalent factor H binding protein meningococcal serogroup B vaccine elicits bactericidal antibodies against representative non-serogroup B meningococci.

MenB-FHbp (Trumenba®; bivalent rLP2086) is a meningococcal serogroup B vaccine containing 2 variants of the recombinant lipidated factor H binding protein (FHbp) antigen. The expression of FHbp, an outer membrane protein, is not restricted to serogroup B strains of Neisseria meningitidis (MenB). This study investigated whether antibodies elicited by MenB-FHbp vaccination also protect against non-MenB strains. Immunological responses were assessed in serum bactericidal assays using human complement (hSBAs) with non-MenB disease-causing test strains from Europe, Africa, and the United States. Importantly, FHbp variant distribution varies among meningococcal serogroups; therefore, strains that code for serogroup-specific prevalent variants (ie, representative of the 2 antigenically distinct FHbp subfamilies, designated subfamily A and subfamily B) and with moderate levels of FHbp surface expression were selected for testing by hSBA. After 2 or 3 doses of MenB-FHbp, 53% to 100% of individuals had bactericidal responses (hSBA titers ≥ 1:8) against meningococcal serogroup C, W, Y, and X strains, and 20% to 28% had bactericidal responses against serogroup A strains; in fact, these bactericidal responses elicited by MenB-FHbp antibodies against non-MenB strains, including strains associated with emerging disease, were greater than the serological correlate of protection for meningococcal disease (ie, hSBA titers ≥ 1:4). This is in comparison to a quadrivalent polysaccharide conjugate vaccine, MCV4 (Menactra®, targeting meningococcal serogroups A, C, W, and Y), which elicited bactericidal responses of 90% to 97% against the serogroup A, C, W, and Y strains and had no activity against serogroup X. Together, these results provide clinical evidence that MenB-FHbp may protect against meningococcal disease regardless of serogroup.

Plasma concentrations of second-line antituberculosis drugs in relation to minimum inhibitory concentrations in multidrug-resistant tuberculosis patients in China: a study protocol of a prospective observational cohort study.

INTRODUCTION: Individualised treatment through therapeutic drug monitoring (TDM) may improve tuberculosis (TB) treatment outcomes but is not routinely implemented. Prospective clinical studies of drug exposure and minimum inhibitory concentrations (MICs) in multidrug-resistant TB (MDR-TB) are scarce. This translational study aims to characterise the area under the concentration-time curve of individual MDR-TB drugs, divided by the MIC for Mycobacterium tuberculosis isolates, to explore associations with markers of treatment progress and to develop useful strategies for clinical implementation of TDM in MDR-TB. METHODS AND ANALYSIS: Adult patients with pulmonary MDR-TB treated in Xiamen, China, are included. Plasma samples for measure of drug exposure are obtained at 0, 1, 2, 4, 6, 8 and 10 hours after drug intake at week 2 and at 0, 4 and 6 hours during weeks 4 and 8. Sputum samples for evaluating time to culture positivity and MIC determination are collected at days 0, 2 and 7 and at weeks 2, 4, 8 and 12 after treatment initiation. Disease severity are assessed with a clinical scoring tool (TBscore II) and quality of life evaluated using EQ-5D-5L. Drug concentrations of pyrazinamide, ethambutol, levofloxacin, moxifloxacin, cycloserine, prothionamide and para-aminosalicylate are measured by liquid chromatography tandem-mass spectrometry and the levels of amikacin measured by immunoassay. Dried blood spot on filter paper, to facilitate blood sampling for analysis of drug concentrations, is also evaluated. The MICs of the drugs listed above are determined using custom-made broth microdilution plates and MYCOTB plates with Middlebrook 7H9 media. MIC determination of pyrazinamide is performed in BACTEC MGIT 960. ETHICS AND DISSEMINATION: This study has been approved by the ethical review boards of Karolinska Institutet, Sweden and Fudan University, China. Informed written consent is given by participants. The study results will be submitted to a peer-reviewed journal. TRIAL REGISTRATION NUMBER: NCT02816931; Pre-results.

The Mla Pathway Plays an Essential Role in the Intrinsic Resistance of Burkholderia cepacia Complex Species to Antimicrobials and Host Innate Components.

Antibiotic resistance is a threat to our modern society, and new strategies leading to the identification of new molecules or targets to combat multidrug-resistant pathogens are needed. Species of the genus Burkholderia, including the Burkholderia cepacia complex (Bcc), Burkholderia pseudomallei, and Burkholderia mallei, can be highly pathogenic and are intrinsically resistant to multiple classes of antibiotics. Bcc species are nonetheless sensitive to extracellular products released by Pseudomonas aeruginosa in interspecies competition. We screened for Burkholderia transposon mutants with increased sensitivity to P. aeruginosa spent medium and identified multiple mutants in genes sharing homology with the Mla pathway. Insertional mutants in representative genes of the Bcc Mla pathway had a compromised cell membrane and were more sensitive to various extracellular stresses, including antibiotics and human serum. More precisely, mla mutants in the Bcc species Burkholderia cenocepacia and Burkholderia dolosa were more susceptible to Gram-positive antibiotics (i.e., macrolides and rifampin), fluoroquinolones, tetracyclines, and chloramphenicol. Genetic complementation of mlaC insertional mutants restored cell permeability and resistance to Gram-positive antibiotics. Importantly, Bcc mla mutants were not universally weaker strains since their susceptibilities to other classes of antibiotics were unaffected. Although cell permeability of homologous mla mutants in Escherichia coli or P. aeruginosa was also impaired, they were not more sensitive to Gram-positive antibiotics or other antimicrobials as was observed in Bcc mla mutants. Together, the data suggest that the Mla pathway in Burkholderia may play a different biological role, which could potentially represent a Burkholderia-specific drug target in combination therapy with antibiotic adjuvants.IMPORTANCE The outer membrane of Gram-negative bacteria acts as an effective barrier against toxic compounds, and therefore compromising this structure could increase sensitivity to currently available antibiotics. In this study, we show that the Mla pathway, a system involved in maintaining the integrity of the outer membrane, is genetically and functionally different in Burkholderia cepacia complex species compared to that in other proteobacteria. Mutants in mla genes of Burkholderia cenocepacia or Burkholderia dolosa were sensitive to Gram-positive antibiotics, while this effect was not observed in Escherichia coli or Pseudomonas aeruginosa The Mla pathway in Burkholderia species may represent an ideal genus-specific target to address their intrinsic antimicrobial resistances.

Validation of a Host Response Assay, SeptiCyte LAB, for Discriminating Sepsis from Systemic Inflammatory Response Syndrome in the ICU.

RATIONALE: A molecular test to distinguish between sepsis and systemic inflammation of noninfectious etiology could potentially have clinical utility. OBJECTIVES: This study evaluated the diagnostic performance of a molecular host response assay (SeptiCyte LAB) designed to distinguish between sepsis and noninfectious systemic inflammation in critically ill adults. METHODS: The study employed a prospective, observational, noninterventional design and recruited a heterogeneous cohort of adult critical care patients from seven sites in the United States (n = 249). An additional group of 198 patients, recruited in the large MARS (Molecular Diagnosis and Risk Stratification of Sepsis) consortium trial in the Netherlands ( www.clinicaltrials.gov identifier NCT01905033), was also tested and analyzed, making a grand total of 447 patients in our study. The performance of SeptiCyte LAB was compared with retrospective physician diagnosis by a panel of three experts. MEASUREMENTS AND MAIN RESULTS: In receiver operating characteristic curve analysis, SeptiCyte LAB had an estimated area under the curve of 0.82-0.89 for discriminating sepsis from noninfectious systemic inflammation. The relative likelihood of sepsis versus noninfectious systemic inflammation was found to increase with increasing test score (range, 0-10). In a forward logistic regression analysis, the diagnostic performance of the assay was improved only marginally when used in combination with other clinical and laboratory variables, including procalcitonin. The performance of the assay was not significantly affected by demographic variables, including age, sex, or race/ethnicity. CONCLUSIONS: SeptiCyte LAB appears to be a promising diagnostic tool to complement physician assessment of infection likelihood in critically ill adult patients with systemic inflammation. Clinical trial registered with www.clinicaltrials.gov (NCT01905033 and NCT02127502).

Four-year antibody persistence and response to a booster dose of a pentavalent MenABCWY vaccine administered to healthy adolescents and young adults.

This open-label, multicenter extension study (NCT02451514) assessed persistence of Neisseria meningitidis serogroups ABCWY antibodies 4 years after primary vaccination. Adolescents and young adults who previously received 2 doses of MenABCWY+OMV (Group III), 1 dose of MenACWY-CRM (Group VI), or newly-recruited vaccine-naïve participants (Group VII) were administered 1 (Group III) or 2 doses (Groups VI and VII) of MenABCWY+OMV, 1 month apart. Immunogenicity was assessed by human serum bactericidal assay (hSBA). Safety and reactogenicity were also evaluated. Percentages of participants with hSBA titers ≥8 (serogroups ACWY), ≥5 (serogroup B) and hSBA geometric mean titers (GMTs) were evaluated in all 129 enrolled participants (Group III: 33; Group VI: 46; Group VII: 50). Anti-ACWY antibody concentrations waned over 4 years post-vaccination, but remained above pre-vaccination concentrations. Similarly, levels of antibodies against serogroup B test strains also waned over 4 years post-vaccination, but remained above pre-vaccination concentrations for some strains. MenABCWY+OMV booster induced a robust anamnestic anti-ACWY response in Group III and VI and a good response against serogroup B test strains (≥82%) in Group III. In serogroup B-naïve participants (Groups VI and VII), anti-B responses to 2 doses of MenABCWY+OMV were less homogenous and lower than in Group III. MenABCWY+OMV was reactogenic, but well-tolerated. No safety concerns were identified. These findings indicate that although antibodies against N. meningitidis serogroups ABCWY waned over 4 years post-vaccination, exposure to a MenABCWY+OMV booster dose elicits an anamnestic response in adolescents previously exposed to the same or another multivalent meningococcal vaccine.

Lower antibody functionality in middle-aged adults compared to adolescents after primary meningococcal vaccination: Role of IgM.

INTRODUCTION: Successful vaccination of elderly persons is often hampered by immunological ageing, leaving part of the elderly population vulnerable for infectious diseases. As an alternative, timely vaccinations might be administered at middle-age, before reaching old age. Studies evaluating the immunological fitness of middle-aged adults are warranted. In this study we compared the immunogenicity of a primary meningococcal vaccination in Dutch middle-aged adults with that in adolescents, in order to gain knowledge on the early signs of immune ageing. METHODS: In this study, we compared the antibody responses after a primary meningococcal vaccination between middle-aged adults (50-65years of age, N=204) and adolescents (10-15years of age, N=225). Blood samples were taken pre-, as well as 28days and 1year post-vaccination. Functional antibody titers were measured with the serum bactericidal killing assay using baby rabbit complement (rSBA). Meningococcal polysaccharide (PS) specific IgG and IgM concentrations were determined with a fluorescent bead-based multiplex immunoassay. RESULTS: Lower post-vaccination functional antibody titers against meningococcal group W and Y were observed in the middle-aged adults compared to the adolescents. One year post-vaccination, also a significantly higher proportion of the middle-aged adults possessed an rSBA titer below protection level. A large reduction in post-vaccination IgM concentrations was observed in the middle-aged adults, whereas IgG concentrations were only marginally different between the two age groups. Strong correlations between the post-vaccination rSBA titers and IgM concentrations were found both in the middle-aged adults and the adolescents. CONCLUSION: Although protective antibody titers were initiated after primary meningococcal vaccination in middle-aged adults, antibody functionality was significantly lower as compared to that in adolescents. This difference was mainly caused by lower IgM responses. Our results indicate early signs of immune ageing in middle-aged adults, which is important knowledge for the development of future vaccine strategies to better protect elderly persons against infectious diseases.

Activity of faropenem with and without rifampicin against Mycobacterium tuberculosis: evaluation in a whole-blood bactericidal activity trial.

Background: Faropenem has in vitro activity against Mycobacterium tuberculosis (Mtb) and shows synergy with rifampicin. We tested this in a whole-blood bactericidal activity (WBA) trial. Methods: We randomized healthy volunteers to receive a single oral dose of faropenem (600 mg) with amoxicillin/clavulanic acid (500/125 mg) ( n = 8), rifampicin (10 mg/kg) ( n = 14) or the combination rifampicin + faropenem + amoxicillin/clavulanic acid ( n = 14). Blood was drawn at intervals to 8 h post-dose. Drug levels were measured using LC-tandem MS. WBA was measured by inoculating blood samples with Mtb and estimating the change in bacterial cfu after 72 h. Trial registration: ClinicalTrials.gov (NCT02393586). Results: There was no activity in the faropenem + amoxicillin/clavulanic acid group (cumulative WBA 0.02 Δlog cfu; P = 0.99 versus zero change). There was a suggestion of a trend favouring the rifampicin + faropenem + amoxicillin/clavulanic acid group at 8 h (cumulative WBA -0.19 ±âŸ0.03 and -0.26 ±âŸ0.03 Δlog cfu in the rifampicin and rifampicin + faropenem + amoxicillin/clavulanic acid groups, respectively; P = 0.180), which was significant in the first hour post-dose ( P = 0.032). Faropenem C max and AUC were 5.4 mg/L and 16.2 mg·h/L, respectively, and MIC for Mtb H37Rv was 5-10 mg/L. Conclusions: Faropenem is not active when used alone, possibly due to inadequate plasma levels relative to MIC. However, there was a suggestion of modest synergy with rifampicin that may merit further testing in clinical trials.

Serologic response to meningococcal vaccination in patients with paroxysmal nocturnal hemoglobinuria (PNH) chronically treated with the terminal complement inhibitor eculizumab.

Eculizumab is indicated for the therapy of patients with symptomatic paroxysmal nocturnal hemoglobinuria (PNH). Due to inhibition of terminal complement cascade, patients on eculizumab are susceptible to Neisseria meningitidis infections. The two mainstays to reduce the risk of infection are vaccination and antibiotic prophylaxis. In this retrospective study, serologic response was analyzed after vaccination with a meningococcal vaccine in 23 PNH patients (median age 36 years; range 25 - 88 years; 15 males, 8 females) by measuring serum bactericidal assay (SBA) using rabbit complement (rSBA) titers against meningococcal serogroups A, C, W, and Y. Serologic protection was defined by an rSBA titer ≥1:8. Forty-three percent (10/23) were vaccinated more than once due to chronic eculizumab treatment. Overall serologic response for the meningococcal serogroups was A: 78% (18/23), C: 87% (20/23), W: 48% (11/23), and Y: 70% (16/23). No meningococcal infections have been observed. As immunological response to vaccines varies, the use of serologic response analyses is warranted. Re-vaccination with a tetravalent conjugate vaccine under eculizumab therapy every 3 years is essential or should be based on response rates. If meningococcal infection is suspected, standby therapy with ciprofloxacin and immediate medical evaluation are recommended. The novel vaccines covering serogroup B may even further reduce the risk for infection.

Demographic and temporal variations in immunity and condition of polar bears (Ursus maritimus) from the southern Beaufort Sea.

Assessing the health and condition of animals in their natural environment can be problematic. Many physiological metrics, including immunity, are highly influenced by specific context and recent events to which researchers may be unaware. Thus, using a multifaceted physiological approach and a context-specific analysis encompassing multiple time scales can be highly informative. Ecoimmunological tools in particular can provide important indications to the health of animals in the wild. We collected blood and hair samples from free-ranging polar bears (Ursus maritimus) in the southern Beaufort Sea and examined the influence of sex, age, and reproductive status on metrics of immunity, stress, and body condition during 2013-2015. We examined metrics of innate immunity (bactericidal ability and lysis) and stress (hair cortisol, reactive oxygen species, and oxidative barrier), in relation to indices of body condition considered to be short term (urea to creatinine ratio; UC ratio) and long term (storage energy and body mass index). We found the factors of sex, age, and reproductive status of the bear were critical for interpreting different physiological metrics. Additionally, the metrics of body condition were important predictors for stress indicators. Finally, many of these metrics differed between years, illustrating the need to examine populations on a longer time scale. Taken together, this study demonstrates the complex relationship between multiple facets of physiology and how interpretation requires us to examine individuals within a specific context.

Ectoparasites as developmental stressors: Effects on somatic and physiological development.

Developmental stress can alter resource allocation in early life, and in altricial birds with rapid developmental trajectories and high resource demands, nestlings may adjust early resource partitioning to cope with challenging environments. We experimentally manipulated ectoparasite levels in nests and assessed whether ectoparasites affected somatic and physiological development in European starling (Sturnus vulgaris) nestlings. We hypothesized that mites act as developmental stressors in nestlings and predicted that nestlings from infested nests would exhibit either reduced somatic growth, or reduced physiological development, including impaired innate immunity, and would have elevated corticosterone concentrations. We either added ≈200 mites to nests during early incubation, or treated nests with a pesticide, permethrin, to reduce mites and possibly other arthropods. We assessed treatment effects on egg spottiness and mite abundance, and monitored offspring hatching and survival. We also measured somatic growth (mass, tarsus length, and feather growth), hematocrit, immune-related metrics (bacterial killing ability [BKA] and spleen mass), and baseline corticosterone concentrations in response to treatment. Compared with mite treatment, permethrin reduced egg spottiness and mite abundance in nests. Relative to nestlings in mite-reduced nests, nestlings in mite-enhanced nests had lower survival, hematocrit, and corticosterone concentrations. Early in development, nestlings from both treatments exhibited similar rapid somatic growth, yet mite-treated nestlings exhibited lower BKA. Nestlings in both treatments increased BKA across development, despite nestlings in mite-treated nests exhibiting lower mass as nest leaving neared. Overall, we found evidence that mites can act as development stressors, but contrary to our prediction, mites decreased corticosterone concentrations.

Repeatability and sources of variation of the bacteria-killing assay in the common snapping turtle.

Research on reptile ecoimmunology lags behind that on other vertebrates, despite the importance of such studies for conservation and evolution. Because the innate immune system is highly conserved across vertebrate lineages, assessments of its performance may be particularly useful in reptiles. The bacteria-killing assay requires a single, small blood sample and quantifies an individual's ability to kill microorganisms. The assay's construct validity and interpretability make it an attractive measure of innate immunity, but it requires proper optimization and sample storage. We optimized this assay for the common snapping turtle (Chelydra serpentina) to assess the repeatability of the assay and the effects of freezing and thawing on bactericidal capacity. We determined whether age (adult female and hatchlings) or incubation temperature influenced bactericidal capacity. We found that the assay was repeatable and that freezing plasma samples for 6 weeks at -80°C did not decrease bactericidal capacity nor did a single 30-min thaw and subsequent refreezing. However, we detected subtle interassay variation and results from one assay were 5-6% greater than those from the other two. Adult females had significantly greater bactericidal ability than hatchlings and we found no relationship between incubation temperature and bactericidal capacity. This assay is a useful tool in snapping turtles and may have applicability in other reptiles. However, species-specific optimization is required to ensure that variation among individuals exceeds interassay variation. Consideration should be given to optimization conditions that facilitate comparisons between or within groups, particularly groups that differ considerably in bactericidal capacity.

The arcA gene contributes to the serum resistance and virulence of Haemophilus parasuis serovar 13 clinical strain EP3.

As a global transcriptional factor, ArcA regulates the expression of hundreds of genes involved in aerobic and anaerobic metabolism. Here we deleted arcA gene and investigated the biological characteristics of arcA deletion mutant (ΔarcA) in Haemophilus parasuis (H. parasuis) serovar 13 clinical strain EP3. Results indicated that deletion of arcA impaired growth of EP3 strain under anaerobic condition, and reduced virulence of EP3 strain in mice. Additionally, the ΔarcA strain showed greater sensitivity in porcine serum and produced less biofilm mass than the EP3 strain. Taken together, these findings suggested that the arcA gene may be involved in pathogenesis in Haemophilus parasuis.

Activity of nitazoxanide and tizoxanide against Mycobacterium tuberculosis in vitro and in whole blood culture.

Nitazoxanide (NTZ) and its metabolite tizoxanide (TIZ) were studied as antimycobacterial agents in vitro (in mycobacterial growth indicator tube [MGIT] cultures) and in a whole blood bactericidal assay. Both NTZ and TIZ show high protein binding. In MGIT cultures (albumin concentration = 78 µM), inhibition of Mycobacterium tuberculosis growth occurred at total drug concentrations of ≥16 µg/ml, whereas in whole blood cultures (albumin concentration = 350 µM), ≥128 µg/ml was required. Free drug fractions at these two conditions were estimated to be 69% and 2%, respectively. Co-incubation of NTZ and TIZ in human plasma for 72 h nearly completely eliminated their ability to inhibit mycobacterial growth in MGIT. Interactions with plasma proteins may limit the potential of NTZ and TIZ as drugs for human tuberculosis.

Staphylococcus aureus avirulent mutant vaccine induces humoral and cellular immune responses on pregnant heifers.

Bovine mastitis produces economic losses, attributable to the decrease in milk production, reduced milk quality, costs of treatment and replacement of animals. A successful prophylactic vaccine against Staphylococcus aureus should elicit both humoral and cellular immune responses. In a previous report we evaluated the effectiveness of a live vaccine to protect heifers against challenge with a virulent strain. In the present study the immunological response of heifers after combined immunization schedule was investigated. In a first experimental trial, heifers were vaccinated with 3 subcutaneous doses of avirulent mutant S. aureus RC122 before calving and one intramammary dose (IMD) after calving. Antibodies concentration in blood, bactericidal effect of serum from vaccinated animals and lymphocyte proliferation was determined. The levels of total IgG, IgG1 and IgG2 in colostrum and the lymphocyte proliferation index were significantly higher in vaccinated respect to non-vaccinated group throughout the experiment. The second trial, where animals were inoculated with different vaccination schedules, was carried out to determine the effect of the IMD on the level of antibodies in blood and milk, cytokines (IL-13 and IFN-γ) concentration and milk's SCC and bacteriology. The bacterial growth of the S. aureus strains was totally inhibited at 1-3×10(6) and 1-3×10(3)cfu/ml, when the strains were mixed with pooled serum diluted 1/40. The results shown that IMD has not a significant effect on the features determinate. In conclusion, a vaccination schedule involving three SC doses before calving would be enough to stimulate antibodies production in milk without an IMD. Furthermore, the results showed a bactericidal effect of serum from vaccinated animals and this provides further evidence about serum functionality. Immune responses, humoral (antigen-specific antibodies and Th2 type cytokines) and cellular (T-lymphocyte proliferation responses and Th1 type cytokines), were augmented by administration of the avirulent mutant which represent an antigenic pool.