Temperature-induced changes in egg white antimicrobial concentrations during pre-incubation do not influence bacterial trans-shell penetration but do affect hatchling phenotype in Mallards

Microbiome formation and assemblage are essential processes influencing proper embryonal and early-life development in neonates. In birds, transmission of microbes from the outer environment into the egg’s interior has been found to shape embryo viability and hatchling phenotype. However, microbial transmission may be affected by egg-white antimicrobial proteins (AMPs), whose concentration and antimicrobial action are temperature-modulated. As both partial incubation and clutch covering with nest-lining feathers during the pre-incubation period can significantly alter temperature conditions acting on eggs, we experimentally investigated the effects of these behavioural mechanisms on concentrations of both the primary and most abundant egg-white AMPs (lysozyme and avidin) using mallard (Anas platyrhychos) eggs. In addition, we assessed whether concentrations of egg-white AMPs altered the probability and intensity of bacterial trans-shell penetration, thereby affecting hatchling morphological traits in vivo. We observed higher concentrations of lysozyme in partially incubated eggs. Clutch covering with nest-lining feathers had no effect on egg-white AMP concentration and we observed no association between concentration of egg-white lysozyme and avidin with either the probability or intensity of bacterial trans-shell penetration. The higher egg-white lysozyme concentration was associated with decreased scaled body mass index of hatchlings. These outcomes demonstrate that incubation prior to clutch completion in precocial birds can alter concentrations of particular egg-white AMPs, though with no effect on bacterial transmission into the egg in vivo. Furthermore, a higher egg white lysozyme concentration compromised hatchling body condition, suggesting a potential growth-regulating role of lysozyme during embryogenesis in precocial birds.

761. Host Intestinal Defenses Against Clostridioides difficile Infection in Chemotherapy Patients

Background Clostridioides difficile infection (CDI) is a common complication in patients undergoing cancer treatment with cytotoxic chemotherapy. Exposure to antibiotics or chemotherapy disrupts the microbiome by killing protective intestinal flora which consequently promotes C. difficile spore germination and disease. The host defense against CDI includes colonization resistance conferred by the healthy microbiome and innate defenses provided by intestinal epithelial cells. One protective factor secreted by Paneth cells of the intestinal epithelium is lysozyme, an enzyme that degrades the cell walls of Gram-positive bacteria such as C. difficile. We hypothesized that chemotherapy-induced mucosal barrier injury and the resultant death of Paneth cells leads to decreased production of lysozyme. We thus sought to examine changes in lysozyme concentration in stools of chemotherapy patients. Methods We collected stool samples from six patients undergoing cancer treatment at four different time points. The first stool sample corresponded to the day prior to the start of chemotherapy (day zero). We then performed ELISA assays to determine the lysozyme concentration for each stool sample. Results On day zero, the lysozyme levels (n=6) averaged 268.1 ± 131.7 ng/mL. Over the course of chemotherapy, the lysozyme levels decreased 78.70 ± 24.19% from the starting value. The lowest values were observed around days 5 through 11 for most patients, coinciding with when they were most neutropenic around day 11. One of the patients developed CDI on day 5 and experienced more fluctuating lysozyme levels thereafter. On the day that the patient developed CDI, lysozyme was measured as 6.63 ng/mL. Throughout treatment, 3/6 patients showed recovery of lysozyme production with white blood cell recovery. Conclusion Our data indicate that chemotherapy causes decreased concentrations of lysozyme in stool. Low lysozyme levels could in part account for the increased susceptibility to CDI during chemotherapy. Future experiments will include bioinformatics analyses to determine how the microbiome changes in response to chemotherapy. Together, these experiments will inform our approach to determining patient susceptibility to chemotherapy-associated CDI. Disclosures All Authors: No reported disclosures

Molecularly Imprinted Magnetic Fluorescent Nanocomposite-Based Sensor for Selective Detection of Lysozyme

A new strategy for the design and construction of molecularly imprinted magnetic fluorescent nanocomposite-based-sensor is proposed. This multifunctional nanocomposite exhibits the necessary optics, magnetism and biocompatibility for use in the selective fluorescence detection of lysozyme. The magnetic fluorescent nanocomposites are prepared by combining carboxyl- functionalized Fe3O4 magnetic nanoparticles with l-cysteine-modified zinc sulfide quantum dots (MNP/QDs). Surface molecular imprinting technology was employed to coat the lysozyme molecularly imprinted polymer (MIP) layer on the MNP/QDs to form a core-shell structure. The molecularly imprinted MNP/QDs (MNP/QD@MIPs) can rapidly separate the target protein and then use fluorescence sensing to detect the protein; this reduces the background interference, and the selectivity and sensitivity of the detection are improved. The molecularly imprinted MNP/QDs sensor presented good linearity over a lysozyme concentration range from 0.2 to 2.0 μM and a detection limit of 4.53 × 10−3 μM for lysozyme. The imprinting factor of the MNP/QD@MIPs was 4.12, and the selectivity coefficient ranged from 3.19 to 3.85. Furthermore, the MNP/QD@MIPs sensor was applied to detect of lysozyme in human urine and egg white samples with recoveries of 95.40–103.33%. Experimental results showed that the prepared MNP/QD@MIPs has potential for selective magnetic separation and fluorescence sensing of target proteins in biological samples.

Partial incubation-induced changes in concentrations of egg white antimicrobials do not influence trans-shell infection but affect hatchling phenotype

Host-microbiome interactions during embryonal and early phase of life is critical point in microbiome formation and assemblage in neonates. In birds, transmission of microbes from the outer environment into the egg interior was found to shape embryo viability and hatchlings phenotype. Microbes’ transmission may be modulated by egg white antimicrobial proteins (AMPs) whose concentration and antimicrobial action are temperature-modulated. As partial incubation and clutch covering with nest-lining feathers during pre-incubation period may both significantly alter temperature conditions acting on eggs, we experimentally investigated effects of these behavioural mechanisms on the concentrations of primary egg white AMPs - lysozyme and avidin using Mallard (Anas platyrhychos) eggs. Moreover, we studied in vivo if concentrations of egg white AMPs reduced probability and intensity of bacterial trans-shell infection and hatchlings phenotype. We found significantly higher egg white lysozyme concentration, while avidin concentration tended to be higher in partially incubated eggs. Clutch covering with nest-lining feathers had no effect on egg white AMPs concentrations. Neither probability nor intensity of bacterial trans-shell infection was associated with concentrations of egg white AMPs. Finally, increased egg white lysozyme was associated with decreased scaled body mass index of hatchlings. These outcomes demonstrate that incubation prior to clutch completion in precocial birds may modulate concentrations of particular egg white AMPs, yet without any effect on transmission of bacteria into the egg in vivo. Furthermore, increased egg white lysozyme may compromise body condition of hatchlings supporting growth-regulating role of lysozyme during embryogenesis in precocial birds.

Ocular Surface Pathology in Patients Suffering From Mercury Intoxication Is Consistent With Neurogenic Dry Eye Disease

Purpose: To report ocular surface pathology of patients with acute/subacute mercury intoxication.Methods: Male workers intoxicated with inorganic mercury were examined for dry eye (DE)-related symptoms. Examinations included Ocular Surface Disease Index questionnaire; tear osmolarity, tear break-up time (T-BUT) and production; mechanical and thermal corneal sensitivity; corneal nerve and dendritic cell density analysis; and analysis of 23 tear cytokines.Results: Most patients, 63.6%, had severe DE-related symptoms. Tear osmolarity was elevated in 83.4%, and T-BUT was low in 22.7% of patients. Tear production and tear lysozyme concentration were low in 13.6% and 27.3% of cases, respectively. Corneal sensitivity thresholds for mechanical, heat and cold stimuli were higher than controls. Densities of nerves, nerve branching, and dendritic cells were lower than in controls. Patient tear levels of IL-12p70, IL1-RA, RANTES, and VEGF were increased, whereas EGF, IL-6, and IP-10/CXCL10 were decreased. Based on cytokine levels, two clusters of patients were identified. Cluster 2 patients had significantly increased tear levels of 18 cytokines, decreased tear lysozyme, lower nerve branching density, fewer dendritic cells, and higher urine mercury levels.Conclusions: Mercury poisoning produced previously undescribed ocular surface pathology, similar to neurogenic inflammatory type of DE and different from the more common DE subtypes.

The impact of parturition induction with cloprostenol on immunological parameters in the sow colostrum

Background Farrowing induction with prostaglandin F2 analogue cloprostenol is commonly used on commercial farms to manage the timing of farrowing. When labour induction is applied, the questions arise about possible side effects of such a hormonal intervention on physiological processes connected with labour and lactation, including colostral immunity. Results In this study, immune cells composition, lysozyme concentration, complement bacteriolytic activity and proinflamatory (GM-CSF2, IL-1β, IL-6, a TNFα) and anti-inflammatory (IL-4, IL-10, TGFβ1 a TGFβ2) cytokines were measured in colostrum samples from sows farrowing naturally (NP) and from sows with farrowing induced using cloprostenol administration on day 113 of gestation (IP). A significantly higher proportion of lymphocytes was found in colostrum of induced sows compared to colostrum of non-induced sows. No significant differences between NP and IP were found in complement activity, in the proportions of granulocytes, macrophages and lymphocyte subpopulations. Lower lysozyme concentration and higher IL-1β, IL-6, TGFβ1 and TNFα concentrations were found in IP sow colostrum compared to colostrum from NP sows. Conclusions An increased proportion of colostral lymphocytes can positively influence the cellular immunity transmission from sow to her offspring. On the other hand, a lower lysozyme concentration can adversely affect newborn’s intestinal immunity, as well as changes in cytokine concentrations can have an adverse effect on newborn piglet intestinal epithelium development and its defence function.

The impact of parturition induction with cloprostenol on immunological parameters in the sow colostrum

Background: Farrowing induction with prostaglandin F2 analogue cloprostenol is commonly used on commercial farms to manage the timing of farrowing. When labour induction is applied, the questions arise about possible side effects of such a hormonal intervention on physiological processes connected with labour and lactation, including colostral immunity. Results: In this study, immune cells composition, lysozyme concentration, complement bacteriolytic activity and proinflamatory (GM-CSF2, IL-1β, IL-6, a TNFα) and anti-inflammatory (IL-4, IL-10, TGFβ1 a TGFβ2) cytokines were measured in colostrum samples from sows farrowing naturally (NP) and from sows with farrowing induced using cloprostenol administration on day 113 of gestation (IP). A significantly higher proportion of lymphocytes was found in colostrum of induced sows compared to colostrum of non-induced sows. No significant differences between NP and IP were found in complement activity, in the proportions of granulocytes, macrophages and lymphocyte subpopulations. Lower lysozyme concentration and higher IL-1β, IL-6, TGFβ1 and TNFα concentrations were found in IP sow colostrum compared to colostrum from NP sows. Conclusions: An increased proportion of colostral lymphocytes can positively influence the cellular immunity transmission from sow to her offspring. On the other hand, a lower lysozyme concentration can adversely affect newborn’s intestinal immunity, as well as changes in cytokine concentrations can have an adverse effect on newborn piglet intestinal epithelium development and its defence function.

The Impact of Parturition Induction With Cloprostenol on Immunological Parameters in the Sow Colostrum

Background: Farrowing induction with prostaglandin F2 analogue cloprostenol is commonly used on commercial farms to manage the timing of farrowing. When labour induction is applied, the questions arise about possible side effects of such a hormonal intervention on physiological processes connected with labour and lactation, including colostral immunity.Results: In this study, immune cells composition, lysozyme concentration, complement bacteriolytic activity and proinflamatory (GM-CSF2, IL-1β, IL-6, a TNFα) and anti-inflammatory (IL-4, IL-10, TGFβ1 a TGFβ2) cytokines were measured in colostrum samples from sows farrowing naturally (NP) and from sows with farrowing induced using cloprostenol administration on day 113 of gestation (IP). A significantly higher proportion of lymphocytes was found in colostrum of induced sows compared to colostrum of non-induced sows. No significant differences between NP and IP were found in complement activity, in the proportions of granulocytes, macrophages and lymphocyte subpopulations. Lower lysozyme concentration and higher IL-1β, IL-6, TGFβ1 and TNFα concentrations were found in IP sow colostrum compared to colostrum from NP sows. Conclusions: An increased number of colostral lymphocytes can positively influence the cellular immunity transmission from sow to her offspring. On the other hand, a lower lysozyme concentration can adversely affect newborn’s intestinal immunity, as well as changes in cytokine concentrations can have an adverse effect on newborn piglet intestinal epithelium development and its defence function.

Sensitivity Enhancement in the Colorimetric/Spectroscopic Determination of Lysozyme Concentration in Nanomolar Level with Colloidal Citrate Capped Au@Ag Core-Shell Nanoparticles

Background: Lysozyme level in body fluids is a significant indicator of various diseases. Cheap and simple colorimetric detection of lysozyme, in biological sample, by gold and silver nanoparticles is a field of interest of nanoparticle research for more than a decade Objective: We report here an attempt to improve the sensitivity part of the colorimetric lysozyme detection process by using citrate capped gold-silver core-shell nanoparticles without any functionalization Methods: Performance of gold-silver core-shell nanoparticles in determining of lysozyme concentration in water has been done using colorimetric/spectroscopic technique. Studies have also been done with gold nanoparticles, silver nanoparticles and mixed colloidal suspension of gold and silver nanoparticles, for comparison. Selectivity study has been done through spectroscopic analysis, red-green-blue colour component analysis and fractal dimension analysis of the nanoparticles interacted with several low and high isoelectric proteins Results: Gold-silver core-shell nanoparticles showed higher sensitivity for a wider range of lysozyme concentration compared to gold and silver nanoparticles prepared by us or reported in literatures. Gold nanoparticles showed higher sensitivity compared to the core-shell nanoparticles, but for a narrow concentration range of lysozyme. For silver nanoparticles and the mixed nanoparticle system, both sensitivity and range of determination of lysozyme concentration were much smaller compared to the core-shell nanoparticles. Core-shell nanoparticles showed better selectivity compared to gold nanoparticles in identifying aquatic solution of lysozyme from that of other proteins Conclusion: Gold-silver core-shell nanoparticles have higher sensitivity in determining wide range of lysozyme concentration in water compared to gold and silver nanoparticles reported in literatures

Livoneca redmanii (Isopoda, Cymothoidae) in meagre Argyrosomus regius: parasitological and molecular diagnosis and proposed control measure

Isopodiosis in cultured meagre Argyrosomus regius was investigated at 3 farms in the northern lakes of Egypt throughout 2018, based upon prevalence rate, parasitological examination, and molecular identification by PCR targeting the large ribosomal subunit 16S of the rRNA gene. Further, the susceptibility of A. regius to isopod infection was experimentally evaluated under hyposalination of 25, 15, and 8 ppt for 1 wk. The isolated isopod stages were morphologically identified as Livoneca redmanii Leach, 1818 with prevalence rates of 77.05 and 77.9% in Al-Madiyyah and Sidi Krir, respectively. The highest prevalence and salinity of 78.85% and 30-34 ppt were reported in El Matareya. DNA sequencing and molecular analysis confirmed the identification of L. redmanii. A. regius experimentally infected with L. redmanii under a hyposalination protocol at 15 and 8 ppt showed marked reduction in mortality (20 and 50%, respectively), and infection prevalence (40 and 63.33%, respectively). The serum lysozyme concentration and nitric oxide of treated fish at 15 and 8 ppt were significantly increased compared to those held at 25 and 33 ppt, whereas serum osmolality levels were higher at 25 and 33 ppt. To our knowledge, this is the first molecular characterization of L. redmanii in cultured A. regius in Egypt.