24-h Post-inoculation Research Articles

Development and validation of a colorimetric antifungal susceptibility testing method for the dimorphic fungus Talaromyces marneffei.

Antifungal drug resistance is an emerging cause of treatment failure in invasive fungal infections, and antifungal susceptibility testing (AFST) may inform treatment decisions. Currently, there are no established AFST guidelines for Talaromyces marneffei (Tm) or other dimorphic fungi. We developed a colorimetric AFST method using a fluorescent redox indicator alamarBlue, which changes from blue to pink in proportion to cellular metabolic activity. We determined the optimal time for alamarBlue addition to be 24h post-inoculation and for MIC reading to be 72h post-inoculation. Our method allows three ways to determine minimum inhibitory concentration (MIC): visual inspection of color change, optical density, and fluorescence intensity. We validated the assay by determining the MICs for seven antifungals against 32 Tm clinical isolates and assessed the essential agreement (EA) and inter-rater reliability between our alamarBlue and the Clinical Laboratory Standard Institute (CLSI) broth microdilution methods. The MIC ranges (from low to high) were: 0.008-0.025 μg/ml for itraconazole, 0.004-0.13 μg/ml for voriconazole, 0.03-0.13μg/ml for posaconazole, 0.06-0.5µg/ml for flucytosine, 0.5-1µg/ml for amphotericin B, 0.5-4µg/ml for caspofungin, and 0.5-16µg/ml for fluconazole. The EAs were 100% between all three MIC readouts of the alamarBlue method, and 94%-100% between the alamarBlue and CLSI methods. Our alamarBlue method had substantially higher inter-rater agreement and offers a more reliable method that can be standardized across laboratories in both high- and low-resource settings compared to the established CLSI methodology.

Bacterial exclusion and wound healing potential of horizontal platelet-rich fibrin (H-PRF) membranes when compared to 2 commercially available collagen membranes.

The aim of the present study was to compare the barrier function during bacterial invasion and wound healing properties of 3 commonly used membranes including horizontal platelet-rich fibrin (H-PRF) against two commercially available resorbable collagen membranes. H-PRF membranes were prepared by collecting venous blood from 3 healthy volunteers using a 700g for 8-min centrifugation protocol followed by compression into membranes. To evaluate their barrier function, 3 groups (H-PRF membrane, collagen membrane A (Bio-Gide, Geistlich), collagen membrane B (Megreen, Shanxi Ruisheng Biotechnology Co) were placed between an inner chamber and outer chamber and inoculated with S. aureus. At 2h, 24h, and 48h post-inoculation, cultures from the inner and outer chambers were assessed for bacterial CFUs. Then, scanning electron microscope (SEM) was utilized to visualized the morphological destruction by bacteria of the inner and outer surfaces of the membranes. To assess the wound healing properties of each membrane, leachates from each group were applied to human gingival fibroblasts (HGF) and a scratch assay was performed at 24h and 48h. S. aureus showed a minimal bacterial attachment or invasion rate through either collagen membranes at 2h post-inoculation, yet over time demonstrated rapid degradation, especially on the rougher surface. While PRF demonstrated higher number of CFUs after 2h, no significant penetration/degradation of the H-PRF membranes was observed at 24h and 48h in the H-PRF group. Both collagen membranes demonstrated significant morphological changes 48h post-bacterial innoculation, while minimal obvious morphological changes were observed in the H-PRF group. The wound healing assay also demonstrated significantly better wound closure rates in the H-PRF group. H-PRF membranes exhibited better barrier function towards S. aureus over 2days of innoculation and better wound healing ability when compared to two commercially available collagen membranes. This study provides further evidence for the application of H-PRF membranes during guided bone regeneration by minimizing bacterial invasion. Furthermore, H-PRF membranes have significantly better ability to promote wound healing.

Heavy metals assessment of ecosystem polluted with wastewaters and taxonomic profiling of multi-resistant bacteria with potential for petroleum hydrocarbon catabolism in nitrogen-limiting medium.

The coexistence of heavy metals (HMs) and petroleum hydrocarbons (PHs) exacerbates ecotoxicity and impair the drivers of eco-functionalities that stimulate essential nutrients for the productivity of the impacted environment. Profiling the bacteria that stem the ecological impact via HMs sequestration and PHs catabolism with nitrogen fixation is imperative to bioremediation of the polluted sites. The sediment of site that was consistently contaminated with industrial wastewaters was analysed for ecological toxicants and the bacterial strains that combined HMs resistance with PHs catabolism in a nitrogen-limiting system were isolated from the sediment and characterized. The geochemistry of the samples revealed the co-occurrence of the above-benchmark concentrations of HMs with the derivatives of hydrocarbons. Notwithstanding, nickel and mercury (with 5% each of the total metal concentrations in the polluted site) exhibited probable effect concentrations on the biota and thus hazardous to the ecosystem. Approx. 31% of the bacterial community, comprising unclassified Planococcaceae, unclassified Bradyrhizobiaceae, Rhodococcus, and Bacillus species, resisted 160µmol Hg2+ in the nitrogen-limiting system within 24h post-inoculation. The bacterial strains adopt volatilization, and sometimes in combination with adsorption/bioaccumulation strategies to sequester Hg2+ toxicity while utilizing PHs as sources of carbon and energy. Efficient metabolism of petroleum biomarkers (> 87%) and Hg2+ sequestration (≥ 75% of 40µmol Hg2+) displayed by the selected bacterial strains portend the potential applicability of the bacilli for biotechnological restoration of the polluted site.

Abstract 12853: Superiority of Intranasal Over Systemic Administration of a Soluble ACE2 Protein (ACE2 618-DDC-ABD) in SARS-CoV-2 Infected Mice

Introduction and Hypothesis. Our group and others are using soluble ACE2 proteins to combat SARS-CoV-2. We have previously reported prevention of mortality, improvement of lung and kidney injury and reduced viral loads in SARS-CoV-2 infected mice that received a novel soluble ACE2 protein with extended duration of action and increased binding affinity for SARS-CoV-2, termed ACE2 618-DDC-ABD. In our previous study, this protein was administered both intranasally (IN) and intraperitoneally (IP). Here we tested the hypothesis that IN is more protective than IP administration. Methods: Human ACE2 618-DDC-ABD was tested in a BSL-3 facility in mice transgenic for human ACE2 (k18hACE2), infected with 3x10 4 PFU SARS-CoV-2. One hour before viral inoculation, the first protein dose was administered followed by 2 consecutive doses 24h and 48h post viral inoculation, either IN or IP (n=10 each). Weight, clinical score and mortality were monitored. Results: In the vehicle-receiving group (BSA in PBS), mortality was 100% on day 5. By contrast, in the IN-group, mortality was reduced markedly to only 10% on day 5 and 14. In the IP-group, by contrast, mortality was 60% at day 5 and 100% at day 14 (fig). In the IP-group, day 5 clinical scores [median (range)] were worse than in the IN-group [3.0 (2.0) and 1.0 (2.0), respectively, p=0.0032]. Conclusions: This study confirms the dramatic improvement in survival in mice treated with an improved soluble ACE2 protein and demonstrates the superiority of IN over IP administration.

Development of a mouse model of ascending infection and preterm birth.

Microbial invasion of the intraamniotic cavity and intraamniotic inflammation are factors associated with spontaneous preterm birth. Understanding the route and kinetics of infection, sites of colonization, and mechanisms of host inflammatory response is critical to reducing preterm birth risk. This study developed an animal model of ascending infection and preterm birth with live bacteria (E. coli) in pregnant CD-1 mice with the goal of better understanding the process of microbial invasion of the intraamniotic cavity and intraamniotic inflammation. Multiple experiments were conducted in this study. To determine the dose of E. coli required to induce preterm birth, CD-1 mice were injected vaginally with four different doses of E. coli (103, 106, 1010, or 1011 colony forming units [CFU]) in 40 μL of nutrient broth or broth alone (control) on an embryonic day (E)15. Preterm birth (defined as delivery before E18.5) was monitored using live video. E. coli ascent kinetics were measured by staining the E. coli with lipophilic tracer DiD for visualization through intact tissue with an in vivo imaging system (IVIS) after inoculation. The E. coli were also directly visualized in reproductive tissues by staining the bacteria with carboxyfluorescein succinimidyl ester (CFSE) prior to administration and via immunohistochemistry (IHC) by staining tissues with anti-E. coli antibody. Each pup's amniotic fluid was cultured separately to determine the extent of microbial invasion of the intraamniotic cavity at different time points. Intraamniotic inflammation resulting from E. coli invasion was assessed with IHC for inflammatory markers (TLR-4, P-NF-κB) and neutrophil marker (Ly-6G) for chorioamnionitis at 6- and 24-h post-inoculation. Vaginally administered E. coli resulted in preterm birth in a dose-dependent manner with higher doses causing earlier births. In ex vivo imaging and IHC detected uterine horns proximal to the cervix had increased E. coli compared to the distal uterine horns. E. coli were detected in the uterus, fetal membranes (FM), and placenta in a time-dependent manner with 6 hr having increased intensity of E. coli positive signals in pups near the cervix and in all pups at 24 hr. Similarly, E. coli grew from the cultures of amniotic fluid collected nearest to the cervix, but not from the more distal samples at 6 hr post-inoculation. At 24 hr, all amniotic fluid cultures regardless of distance from the cervix, were positive for E. coli. TLR-4 and P-NF-κB signals were more intense in the tissues where E. coli was present (placenta, FM and uterus), displaying a similar trend toward increased signal in proximal gestational sacs compared to distal at 6 hr. Ly-6G+ cells, used to confirm chorioamnionitis, were increased at 24 hr compared to 6 hr post-inoculation and control. We report the development of mouse model of ascending infection and the associated inflammation of preterm birth. Clinically, these models can help to understand mechanisms of infection associated preterm birth, determine targets for intervention, or identify potential biomarkers that can predict a high-risk pregnancy status early in pregnancy.

RNA-Sequencing in Resistant (QL3) and Susceptible (Theis) Sorghum Cultivars Inoculated With Johnsongrass Isolates of Colletotrichum sublineola.

Gene expression was analyzed at 0- and 24-h post-inoculation of two inbred sorghum cultivars known to differ in response to inoculation with Colletotrichum sublineola, the fungal pathogen that causes anthracnose. QL3 is reported to have quantitative resistance, while Theis is susceptible to most pathotypes of the pathogen; RNASeq identified over 3,000 specific genes in both cultivars as showing significant changes in expression following inoculation; in all but one gene, the changes in QL3 and Thies were in the same direction. Many other genes showed significant changes in only one of the two cultivars. Overall, more genes were downregulated than upregulated. Differences in changes in expression levels of a few genes suggested potential roles for the difference in disease response between QL3 and Theis, but did not identify known resistance genes. Gene ontology (GO) and pathway enrichment analysis identified upregulation of 23 transcription factor encoding genes as well as genes involved in the production of secondary metabolites, which are part of a typical host defense reaction.

An Exceptional Broad-Spectrum Nanobiocide for Multimodal and Synergistic Inactivation of Drug-Resistant Bacteria

An Exceptional Broad-Spectrum Nanobiocide for Multimodal and Synergistic Inactivation of Drug-Resistant Bacteria

Weighted Gene Co-Expression Network Coupled with a Critical-Time-Point Analysis during Pathogenesis for Predicting the Molecular Mechanism Underlying Blast Resistance in Rice

BackgroundRice blast, caused by the ascomycete fungus M. oryzae, is one of the most important diseases of rice. Although many blast resistance (R) genes have been identified and deployed in rice varieties, the molecular mechanisms responsible for the R gene-mediated defense responses are yet not fully understood.ResultsIn this study, we used comparative transcriptomic analysis to explore the molecular mechanism involved in Piz-t-mediated resistance in a transgenic line containing Piz-t (NPB-Piz-t) compared to Nipponbare (NPB). Clustering and principal component analysis (PCA) revealed that the time-point at 24-h post inoculation (hpi) was the most important factor distinguishing the four time-points, which consisted of four genes of mitogen-activated protein kinases (MAPKs) signaling pathway, one gene related to WRKY DNA-binding domain containing protein, five pathogenesis-related protein (OsPR1s) genes, and three genes of R proteins involving in the most significant protein-protein interaction (PPI) pathway. Using weighted gene co-expression network analysis (WGCNA) to investigate RNA-seq data across 0, 24, 48, and 72 hpi, nine modules with similar patterns expression pattern (SEP) and three modules with differential expression pattern (DEP) between NPB-Piz-t and NPB across 0, 24, 48, and 72 hpi with KJ201 (referred to as Piz-t-KJ201 and NPB-KJ201) were identified. Among these the most representative SEP green-yellow module is associated with photosynthesis, and DEP pink module comprised of two specific expressed nucleotide-binding domain and leucine-rich repeat (NLR) genes of LOC_Os06g17900 and LOC_Os06g17920 of Pi2/9 homologous, three NLR genes of LOC_Os11g11810, LOC_Os11g11770, and LOC_Os11g11920 which are putatively associated with important agronomic traits, and a B3 DNA binding domain containing protein related genes (LOC_Os10g39190). Knockout of LOC_Os10g39190 via CRISPR-Cas9 resulted in plant death at the seedling stage.ConclusionsThe research suggested that Piz-t and multiple NLR network might play important roles in the regulation of the resistance response in the Piz-t-KJ201 interaction system. The identified genes provide an NLR repository to study the rice-M. oryzae interaction system and facilitate the breeding of blast-resistant cultivars in the future.

Restriction of chronic Escherichia coli urinary tract infection depends upon T cell-derived interleukin-17, a deficiency of which predisposes to flagella-driven bacterial persistence.

Urinary tract infections (UTI) frequently progress to chronicity in infected individuals but the mechanisms of pathogenesis underlying chronic UTI are not well understood. We examined the role of interleukin (IL)-17A in UTI because this cytokine promotes innate defense against uropathogenic Escherichia coli (UPEC). Analysis of UPEC persistence and pyelonephritis in mice deficient in IL-17A revealed that UPEC CFT073 caused infection at a rate higher than the multidrug resistant strain EC958. Il17a-/- mice exhibited pyelonephritis with kidney bacterial burdens higher than those of wild-type (WT) mice. Synthesis of IL-17A in the bladder reflected a combination of γδ-T and TH 17 cell responses. Analysis of circulating inflammatory mediators at 24h postinoculation identified predictors of progression to chronicity, including IL-6 and monocyte chemoattractant protein-1 (MCP-1). Histological analysis identified infiltrating populations of neutrophils, NK cells, and γδ T cells in the bladder, whereas neutrophils predominated in the kidney. Analysis of the contribution of flagella to chronicity using hyper-flagellated and fliC-deficient UPEC in WT and Il17a-/- mice revealed that, in a host that is deficient for the production of IL-17A, flagella contribute to bacterial persistence. These findings show a role for IL-17A in defense against chronic UTI and a contribution of flagella to the pathogenesis of infection.

Clinical observation, acute phase protein levels, and histopathological changes of mammary gland in experimentally infected goats with Staphylococcus aureus

Staphylococcus aureus (S. aureus) is a major cause of mastitis in small ruminants, and studies on the potential application of acute phase proteins (APPs) in disease biomarkers and understanding the disease mechanism in mastitis affecting small ruminants are limited. Thus, the objective of this study was to determine clinical, histopathological changes and serum concentrations of serum amyloid A (SAA) and haptoglobin (Hp) associated with experimentally induced mastitis using S. aureus. Left mammary quarter in the does was challenged with sterile Ringers buffered salt solution as group 1 (control animals; n = 6) and S. aureus (1.5 × 108 cfu/ml) as group 2 (inoculated does; n = 5) respectively. The animals were monitored at various time intervals for the signs of mastitis and serum concentrations of Hp and SAA. Majority of the animals in the inoculated group showed typical signs of acute mastitis characterized with fever, warm, painful, and swollen mammary gland as early as 24-h post-inoculation (PI). There was significant increased (p < 0.05) in the levels of Hp and SAA in the serum of inoculated animals at 24-h post-inoculation. Histopathological changes in the mammary gland of the inoculated goats included generalized and severe infiltration of the interstitial spaces with polymorphonuclear neutrophils (PMN), severe necrosis of secretory cells, and hyperplastic epithelial and luminal lining. In conclusion, inoculation of S. aureus intramammarily leads to an increment of Hp and SAA at 24-h PI which indicates that both APPs played crucial role as biomarkers in detection and monitoring of S. aureus mastitis in goats.

Exploring the TLR and NLR signaling pathway relevant molecules induced by the Theileria annulata infection in calves.

Theileria annulata is the pathogen of bovine tropical theileriosis. It is extremely harmful to the cattle industry, with huge economic losses. The toll-like receptor (TLR) and NOD-like receptor (NLR) signaling pathways are crucial for resistance to infection of the protozoa, such as Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma cruzi. However, the role of these immune-related pathways is unclear during T. annulata infection. In the present study, peripheral blood mononuclear cells and serum were separated from blood samples of calves infected with homogenized tick supernatants carrying T. annulata sporozoites at 12h, 24h, 36h, 48h, 72h, 96h, 120h, 144h and 168h postinoculation. The Custom RT2 Profiler PCR Array was used to explore the mRNA levels of 42 TLR and NLR signaling pathway relevant genes. The TLR1, TLR6, TLR10, NLRP1, and MyD88 genes and their downstream signaling molecules significantly differed after the T. annulata infection in comparison with that of preinfection from 72h to 168h postinoculation. The serum concentrations of IL-6, IL-1β, and TNFα were significantly increased at 96h and 168h postinfection. These findings provided novel information to help determine the mechanisms of TLR and NLR signaling pathway involvement in protection against T. annulata infection.

Comparative proteomics analysis of young spikes of wheat in response to Fusarium graminearum infection

To investigate signaling events and defense responses in wheat–Fusarium graminearum interaction, the proteomes of young spikes with and without 24 h of F. graminearum inoculation were investigated in the resistant wheat variety Sumai 3 and the susceptible Huaimai 27. Totally 96 protein spots displayed differential expressions in one or two materials after inoculation. 65 of them were identified through mass spectrometry analysis. These proteins can be functionally classified into 12 categories and most of them participate in defense response and signaling. Our study revealed an accumulation of defense/stress response proteins, suppression of protein synthesis and photosynthesis, and changes in basal and energy metabolisms during compatible and incompatible interactions. However, differential regulation of hormone signaling, proteasome-mediated proteolysis, nitrogen metabolism, transcription, and protein modification was observed between two interactions. This result was further confirmed by analyzing the corresponding genes of 19 important proteins from six functional groups at the mRNA level. In addition, the resistant and susceptible wheat varieties showed differences in antioxidant enzymes activities and malondialdehyde contents 24-h postinoculation. Overall, the current study allows us to further dissect the possible metabolism and regulation progresses activated in F. graminearum-treated wheat spikes, and offers new understanding of mechanisms underlying wheat scab resistance.

Mechanism of Anti-rotavirus Synergistic Activity by Epigallocatechin Gallate and a Proanthocyanidin-Containing Nutraceutical.

Epigallocatechin gallate (EGCG) of green tea and the nutraceutical CystiCran®-40 (containing 40% proanthocyanidins) of the cranberry plant have been associated with antiviral activity. The purpose of this work was to determine the mechanism of antiviral synergy between each compound. Coliphage T4II (phage T4) and the rotavirus strain SA-11(RTV) were used as model virus systems. Individual and combined flavonoids structural and molecular weight analyses were performed by NMR and HPCL/MS, respectively. A suboptimal concentration of EGCG or C-40 alone or in combination reduced phage infectivity by ≤10%. Similarly, EGCG (30µg/ml) and C-40 (25µg/ml), respectively, reduced RTV titers by 3 and 13%. However, RTV titers were reduced by 32% (p<.05) with both flavonoids used in combination. RTV was not recognized in host cells by electron microscopy 24-h post-inoculation. NMR and HPLC/MS findings revealed significant structural and potential changes in molecular weight of the flavonoids in complex.

The wheat Lr34 multipathogen resistance gene confers resistance to anthracnose and rust in sorghum.

SummaryThe ability of the wheat Lr34 multipathogen resistance gene (Lr34res) to function across a wide taxonomic boundary was investigated in transgenic Sorghum bicolor. Increased resistance to sorghum rust and anthracnose disease symptoms following infection with the biotrophic pathogen Puccinia purpurea and the hemibiotroph Colletotrichum sublineolum, respectively, occurred in transgenic plants expressing the Lr34res ABC transporter. Transgenic sorghum lines that highly expressed the wheat Lr34res gene exhibited immunity to sorghum rust compared to the low‐expressing single copy Lr34res genotype that conferred partial resistance. Pathogen‐induced pigmentation mediated by flavonoid phytoalexins was evident on transgenic sorghum leaves following P. purpurea infection within 24–72 h, which paralleled Lr34res gene expression. Elevated expression of flavone synthase II, flavanone 4‐reductase and dihydroflavonol reductase genes which control the biosynthesis of flavonoid phytoalexins characterized the highly expressing Lr34res transgenic lines 24‐h post‐inoculation with P. purpurea. Metabolite analysis of mesocotyls infected with C. sublineolum showed increased levels of 3‐deoxyanthocyanidin metabolites were associated with Lr34res expression, concomitant with reduced symptoms of anthracnose.

Synthesis and characterization of chitosan nanoparticles and their effect on Fusarium head blight and oxidative activity in wheat

The main aim of present study was to prepare chitosan (CS) and chitosan nanoparticles (CS/NPs) to evaluate their antifungal and oxidative activity. CS/NPs were prepared based on the ionic gelation of CS with tripolyphosphate (TPP) anions by using centrifugation and pH change. The obtained nanoparticles (NPs) were characterized by size and zeta potential analysis. The antifungal activity of the CS and CS/NPs were evaluated on the Fusarium graminearum, which causes Fusarium head blight (FHB) on wheat by the method of spraying on the Potato dextrose agar (PDA) medium. The Dynamic light scattering (DLS) indicated that particle diameter (z-average) was approximately 180.9±35.5–339.4±50.9 and 225.7±42.81–595.7±81.7nm for NPs prepared from CS with different molecular weights by using centrifugation and pH change methods, respectively. Different concentrations of CS and NPs were tested to know the inhibitory effect of F. graminearum. Low molecular weight (LMW) CS and its NPs had high potential of antifungal activity on suppress of fungus growth. The maximum percentage of growth reduction was 68.18%, and 77.5% by CS and its NPs at concentrations of 1000 and 5000ppm, respectively. In greenhouse trials, at 28days after inoculation (dpi), the area under the disease progress curve (AUDPC) from 7 dpi to 28 dpi of control plants treated with acetic acid aqueous solution and distilled water was almost up to 7.36 and 7.7, respectively, while plants treated with CS and NPs only had approximately 3.61 and 3.34, respectively. Results revealed that H2O2 accumulations displayed a different pattern during the activation of plant defense systems, it had brownish sites on the infected palea. Since 24h post inoculation (hpi), the H2O2 accumulations were shown in both CS and NPs, and the elevated H2O2 accumulation appeared in 72 hpi in both treatments. CS and NPs at high concentration increased the degree of tissue and cell injury. The obtained results clearly suggest that CS and its NPs have remarkable potential for further field screening towards crop protection.

Short interfering RNAs targeting a vampire-bat related rabies virus phosphoprotein mRNA.

The aim of this study was to assess the in vitro and in vivo effects of short-interfering RNAs (siRNAs) against rabies virus phosphoprotein (P) mRNA in a post-infection treatment for rabies as an extension of a previous report (Braz J Microbiol. 2013 Nov 15;44(3):879–82). To this end, rabies virus strain RABV-4005 (related to the Desmodus rotundus vampire bat) were used to inoculate BHK-21 cells and mice, and the transfection with each of the siRNAs was made with Lipofectamine-2000™. In vitro results showed that siRNA 360 was able to inhibit the replication of strain RABV-4005 with a 1 log decrease in virus titter and 5.16-fold reduction in P mRNA, 24 h post-inoculation when compared to non-treated cells. In vivo, siRNA 360 was able to induce partial protection, but with no significant difference when compared to non-treated mice. These results indicate that, despite the need for improvement for in vivo applications, P mRNA might be a target for an RNAi-based treatment for rabies.

Edwardsiella tarda induces dynamic changes in immune effector activities and endocrine network of Pangasius pangasius (Hamilton, 1822)

Dynamic changes in immune effector and endocrine regulatory activities of pangas catfish, Pangasius pangasius against Edwardsiella tarda infection were evaluated in this study. Healthy pangas were inoculated intramuscularly with a dose of 2.17×107 cells/fish to induce edwardsiellosis. The challenged pangas exhibited excessive mucus secretion, petechial hemorrhages and protruded hemorrhagic anus after 24h post-inoculation. The non-specific humoral defense factors such as serum lysozyme and anti-protease activity showed a steady increase from 5dpi. A sustained ceruloplasmin activity against E. tarda infection was observed. The non-specific cellular defense factor respiratory oxidative burst activity decreased on 5dpi followed by an increase on 10dpi. The myeloperoxidase activity and nitric oxide production showed an increase on 5dpi followed by reduction. Analysis of specific immune parameter in-vitro revealed suppression of lymphocyte proliferation, when stimulated using E. tarda outer membrane protein. The antibody production, as measured by indirect ELISA, appeared to be suppressed until 15dpi and increased thereafter till the end of the experiment. A robust secondary immune response was observed with an enhanced dose after 10dpi. Further, the level of serum insulin-like growth factor-1 (IGF-1) was reduced significantly when challenged with E. tarda and did not reach the normalcy even after 22dpi. The levels of serum cortisol and insulin-like growth factor binding protein-1 (IGFBP-1) increased significantly on 5dpi and reduced thereafter to near normal on and after 15dpi. The results of this first kinetic study suggest that E. tarda infection may influence the endocrine regulatory and immune effector activities of P. pangasius significantly. Statement of relevance➣Infectious diseases in farmed catfish can result in 60–100% loss.➣Edwardsiella tarda infection in catfish negatively impacts the regulation of GH/IGF-I/IGFBP network and the production.

Infection of Human Retinal Pigment Epithelium with Chlamydia trachomatis.

PurposeLittle is known about the susceptibility of posterior segment tissues, particularly the human retinal pigment epithelium (hRPE), to Chlamydia trachomatis. The purpose of the study was to investigate the possibility of infecting the hRPE with Chlamydia trachomatis, and to examine the infectivity of different Chlamydia trachomatis clinical isolates for hRPE cells and the hRPE cell response to the infection.MethodsCultured hRPE and McCoy cells were inoculated with eight Chlamydia trachomatis (serovar E) clinical isolates at multiplicity of infection (MOI) of 2.0 or 0.3. To detect Chlamydia trachomatis, samples were stained immunohistochemically with anti-major outer membrane protein antibodies at 24h, 48h, and 72h postinoculation (PI). The changes in the expression of signaling molecules and proteins of cytoskeleton and extracellular matrix in hRPE cells were examined immunohistochemically.ResultsAll eight clinical isolates demonstrated ability to infect hRPE cells. At equal MOI of 0.3, the infectivity of Chlamydia trachomatis clinical isolates for RPE culture was found to be at least as high as that for McCoy cell culture. At 24h PI, the percentage of inclusion-containing cells varied from 1.5 ± 0.52 to 14.6 ± 3.3% in hRPE cell culture infected at MOI of 2.0 against 0.37 ± 0.34 to 8.9 ± 0.2% in McCoy cell culture infected at MOI of 0.3. Collagen type I, collagen type IV, basic fibroblast growth factor, transforming growth factor-beta and interleukin–8 expression at 48h PI were maximally increased, by 2.1-, 1.3-, 1.5-, 1.5- and 1.6-fold, respectively, in the Chlamydia trachomatis-infected compared with control hRPE cell culture specimens (P < 0.05).ConclusionsThis study, for the first time, proved the possibility of infecting hRPE cultured cells with Chlamydia trachomatis, which leads to proproliferative and proinflammatory changes in the expression of signaling molecules and extracellular matrix components.

Ex vivo modeling of feline herpesvirus replication in ocular and respiratory mucosae, the primary targets of infection

Feline herpesvirus 1 (FeHV-1) is a major cause of rhinotracheitis and ocular diseases in cats. In the present study, the viral replication at the primary infection sites was studied using feline respiratory and ocular mucosa explants. The explants of three cats were maintained in an air–liquid culture up to 96hours without loss of viability. After inoculation with FeHV-1 (C27), no evidence of infection was noted in corneal epithelium, while plaque-wise replication was observed in conjunctival and tracheal mucosae beginning from 24h post inoculation (hpi). The viral plaque diameters increased over time in trachea and conjunctiva and were larger in tracheal explants than in conjunctival explants at 48hpi. FeHV-1 penetrated the basement membrane in conjunctival and tracheal explants between 24 and 48hpi. At 48 and 72hpi, viral invasion was going deeper in tracheal explants than in conjunctival explants. Our study indicates that FeHV-1 has a better capacity to invade the respiratory mucosa than the conjunctival mucosa, and prefers the conjunctiva, but not the cornea as a portal of entry during ocular infection.

Effect of Zinc Supplementation on Immune Response in Goats

This study was undertaken to evaluate the effects of supplemental zinc (Zn) immune response in growing goats. Twenty-four healthy goat kids were randomly divided into four groups of six kids each. Kids were fed basal ration consisted of concentrate feed mixture and green berseem to meet the nutrient requirements of goats (NRC, 1981). Kids in the group one were fed basal diet which contains 33.02 mg/kg dry mater (DM) zinc from ingredient of ration without any Zn supplement in their minerals mixture. Kids in the two, three and four groups were fed the same basal diet of group one with additive 20, 50 and 100 ppm Zn (as ZnSO4) in their diets, respectively. Amounts of 50 and 100 mg Zn/kg DM have significantly higher humoral immune response at all the three intervals. Skin-fold thickness was significantly higher at 24-h post-inoculation indicating improved cellular immunity with 50 and 100 mg Zn/kg DM. The results showed that a diet containing 83.02 mg Zn/kg DM was adequate to support better cellular and humoral immune response in growing goats.