Primary Inoculation Research Articles

Defence responses triggered by Blumeria graminis f.sp. hordei in non‐host wheat genotypes results in a decrease in Puccinia triticina infection

AbstractNon‐host resistance is the resistance of plants against all so‐called non‐adapted pathogens, and is considered the most durable and efficient immune system of plants. We investigated barley powdery mildew (Blumeria graminis f.sp. hordei, Bgh)‐triggered defence responses in seven wheat genotypes, and the induction of resistance by this non‐host pathogen to challenge infection with leaf rust, Puccinia triticina Eriks (formerly known as Puccinia recondita f.sp. tritici). Bright‐field and ultraviolet microscopy revealed that although the germination of conidia on non‐host wheat leaves was generally lower than on host barley leaves, the 100% germination on Mv Mambó and only 50% germination on Mv 1 wheat genotypes are the two extremes. If we consider only the germinated conidia, papilla formation was the most frequently observed interaction on Mv 2 (90%) and Mv 4 (88%) genotypes. Epidermal cell death was pronounced on Mv Mambó (31%), but rare on Mv 2 (10%) and Mv 4 (11%), and a mesophyll hypersensitive response could even be detected. At sites of penetration attempts accumulation of H2O2 was shown by 3,3′‐diaminobenzidine staining. Ascorbate peroxidase, glutathione S‐transferase and catalase enzyme activities were augmented by 24.5%, 40.4%, and 21.5%, respectively, in wheat leaves 2 days after Bgh infection. Primary inoculation with Bgh, although with high variability, reduced the number of pustules in all wheat genotypes at first and third leaf stages upon inoculation with leaf rust races 12 and 61 by 10%–70% depending on the genotype, leaf stage and rust race.

Systemic antimycotic therapy of patients with mycosis of the oral mucosa and periodontal Сandida etiology

Candidiasis is the most common mycosis. Оral candidiasis occurs in the form of stomatitis, including the so-called prosthetic stomatitis. Somewhat less often, fungi of the genus Candida are detected with gingivitis and periodontitis, manifestations of red lichen planus on the oral mucosa.The purpose of the work: to analyze the results of mycological studies and determine the sensitivity of fungi isolated from the oral cavity in vari-ous oral pathologies to the main antimycotics, as well as the formation on this basis of an algorithm for antimycotic therapy of candidal lesions of the oral mucosa and periodontium.Material and methods. The paper uses the results of studies of patients with candidiasis of the oral mucosa (127 patients), prosthetic stomatitis (136 patients), candida-associated periodontitis (168 patients). Primary inoculation was performed quantitatively on Sabouraud’s medium and duplicated on M1297 chromogenic medium (Himedia Labs, India).To study the biofilm formed on the oral mucosa, fixed preparations of gold-coated scrapings were prepared for scanning electron microscopy, which was performed using a Quanta 200 3D bi-beam microscope (USA). Sensitivity to antimycotics was assessed using the traditional method of disks.The results of the study. When studying the species composition of yeast-like fungi isolated from the material, it was found that the species C. albicans was isolated in 80% of patients, in association with other species: C. Krusei, C. guilliermondii and C. glabrata. The second place was taken by the species C. Krusei, which was detected in 13.3% of patients. Representatives of this genus of yeast fungi are characterized by the formation of a biofilm with a pronounced mantle, which protects the microorganism from aggressive factors, including antimycotics and antiseptics. All the studied preparations showed fungicidal activity against these strains of yeast-like fungi. When registering the results, it was found that the most pronounced sensitivity of yeast-like fungi of this species was to drugs from the group of azole derivatives. The most pronounced antifungal activity was noted by us in voriconazole.Conclusion. Based on the obtained data, an algorithm of systemic antimycotic therapy for oral candidiasis has been developed.

ВИДОВОЙ СОСТАВ АССОЦИАЦИЙ И ВЗАИМООТНОШЕНИЯ МИКРООРГАНИЗМОВ, ВЫДЕЛЕННЫХ ИЗ ОСТЕОМИЕЛИТИЧЕСКОГО ОЧАГА

Objective. To determine the species composition of the associations isolated from osteomyelitis foci and to study the character of associate relationships based on the biofilm-forming ability data. Methods. The microbiological study included clinical isolates (n=184) obtained from associations (n=88) during primary inoculations from wounds and fistulas of patients (n= 88) with chronic osteomyelitis of long tubular bones. In order to obtain an associative biofilm in vitro, the cultures of competing bacterial strains were daily mixed in 1:1 ratio. The biofilms were grown on the surface of polystyrene plates with subsequent determination of the level of biofilm formation in 24 and 48 hours. Thecoefficient of relationship (CR) was calculated to evaluate the synergistic, neutral and antagonistic relationships between bacteria in the biofilms. Results. The associations of staphylococcus with gram-negative bacteria were most frequently recovered from osteomyelitis foci. On the 1st day of the experiment, 38,6 % of associations had a moderate biofilm-forming ability, and besides, associations of gram-positive + gram-negative bacteria were observed in 36,4%; 42,1% of associations had a low biofilm-forming ability; 19,3% - had a high biofilm-forming ability. After 48 hours the percentage of mild adhesive strains remained at the same level - 38,6%, as for the low adhesive ones it decreased to 36,4%, high adhesive - increased up to 25%. Most bacterial associations manifested antagonistic relationships. Synergism in biofilm-formingby the association of S. aureus + P. aeruginosa was observed in 2 cases,while the level of film-forming was high as on the first and the second day of the study. In several associations it transformed from antagonistic to synergistic or neutral relationships. Conclusion. It has ben established that among the identified associations, the largest specific weight falls on the associations of gram-positive + gram-negative bacteria , while S. aureus is one of the most common components. These associations were noted to have high and mild activity of biofilm -forming on the surface of polystyrene plates. Relationships between the microorganisms isolated from osteomyelitis foci in associations, as a rule, are antagonistic. What this paper adds For the first time, the nature of the relationship between microorganisms in the composition of associations isolated from the osteomyelitis focus has been studied on the basis of biofilm-forming ability data. It has been established that in microbiocenosis of the osteomyelitis foci, the greatest specific weight falls on the associations of S. aureus with gram-negative bacteria. These associations have a high and moderate activity of biofilm formation on the surface of polystyrene plates. The relationships between microorganisms isolated from the osteomyelitis focus as part of associations are usually antagonistic.

Global epidemiology of coronavirus disease 2019 and lessons for effective control of this and future pandemics

Barely 1 year after severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first reported in China, the virus has infected approximately 120 million people, caused around 3 million deaths, and adversely affected the global economy. Despite stringent measures to flatten the epidemiologic curve of the pandemic, there have been spikes and waves of the infection in many countries, particularly in the American, European, and Asian continents. This review critically evaluated the global epidemiology of the novel coronavirus disease 2019 (COVID-19) to provide advice on other possible ways of managing the disease as various COVID-19 vaccines are being rolled out. To effectively control this and possible future epidemics/pandemics, there is a need to maintain a healthy balance between saving lives and livelihoods in the implementation of lockdowns. Unwarranted human exposures to animals, particularly pangolins, civet cats, bats, dromedary camels, and other wildlife known to be reservoirs and intermediate hosts of coronaviruses should be prevented. All the identified strains of SARS-CoV-2, including the highly infectious UK and South African variants, should be incorporated in COVID-19 vaccine production; to widen the protection spectrum. Some of the COVID-19 vaccines require primary inoculation, booster vaccination after 2-4 weeks and annual revaccination for adequate immunization against SARS-CoV-2. Survivors of COVID-19 may require only a single vaccine dozing and annual revaccination thereafter. Adoption of One Health approach and the development of globally coordinated active surveillance systems against emerging and reemerging zoonotic viruses are imperative.

Co-Cultivation of Fusarium, Alternaria, and Pseudomonas on Wheat-Ears Affects Microbial Growth and Mycotoxin Production.

Mycotoxigenic fungal pathogens Fusarium and Alternaria are a leading cause of loss in cereal production. On wheat-ears, they are confronted by bacterial antagonists such as pseudomonads. Studies on these groups’ interactions often neglect the infection process’s temporal aspects and the associated priority effects. In the present study, the focus was on how the first colonizer affects the subsequent ones. In a climate chamber experiment, wheat-ears were successively inoculated with two different strains (Alternaria tenuissima At625, Fusarium graminearum Fg23, or Pseudomonas simiae Ps9). Over three weeks, microbial abundances and mycotoxin concentrations were analyzed and visualized via Self Organizing Maps with Sammon Mapping (SOM-SM). All three strains revealed different characteristics and strategies to deal with co-inoculation: Fg23, as the first colonizer, suppressed the establishment of At625 and Ps9. Nevertheless, primary inoculation of At625 reduced all of the Fusarium toxins and stopped Ps9 from establishing. Ps9 showed priority effects in delaying and blocking the production of the fungal mycotoxins. The SOM-SM analysis visualized the competitive strengths: Fg23 ranked first, At625 second, Ps9 third. Our findings of species-specific priority effects in a natural environment and the role of the mycotoxins involved are relevant for developing biocontrol strategies.

Development of a Latex Agglutination Test for Detecting Pathogenic Burkholderia and its Approbation in the Endemic Regions of Vietnam

The aim of the work was development of a monoclonal antibody-based latex agglutination test to identify the causative agent of melioidosis, and the approbation of a freeze-dried experimental preparation for screening of environmental bacterial isolates in Vietnam.Materials and methods. The carriers of specific antibodies were polyacrolein latex particles with active aldehyde groups on the surface. Typical strains of the causative agents of melioidosis and glanders with a full-fledged antigenic structure, as well as the strains Burkholderia thailandensis, Burkholderia cepacia, Pseudomonas aeruginosa, and Pseudomonas putida were used to control the test specificity. The latex agglutination reaction was carried out on plastic Petri dishes with daily bacterial cultures, from which suspensions were prepared at a concentration of 1–2·109 m.c./ml. The results of the reaction were registered visually for 5–8 min using a 4-cross system against a dark background under lighting. The reaction to 3–4 crosses was recorded as positive. Colonies suspected of belonging to pathogenic Burkholderia from primary inoculations were transferred to L-agar with polymyxin B and grown for 36 hours at (37±1) °C. The species of the selected suspicious colonies was determined by multiplex PCR.Results and discussion. With collection strains, latex test demonstrated high sensitivity agglutinating 97.7 % of B. pseudomallei and all B. mallei strains. At the same time, it was negative with B. thailandensis, B. cepacia, P. aeruginos and P. putida. In microbiological screening of bacterial cultures isolated from environmental objects, the latex test had a diagnostic sensitivity of 89.4 %. Using the latex test at the stage of primary screening, it is possible to significantly reduce the time when processing a lot of samples received for analysis, as well as to reduce the consumption of reagents used at the subsequent stages of identification.

Comparative evaluation of immune-responsiveness in indigenous and exotic breeds of chicken

In this research we conducted a comparative study of immune responsive traits for the two exotic chicken breeds viz. Australorp and Cornish with one of the indigenous Kadaknath chicken breeds. Immune response to Sheep Red Blood Cells (SRBC) was measured in terms of Haemagglutination Titre (HA) and Haemagglutination Inhibition titre (HI) at five stages viz; 0 (pre inoculation), 3rd , 6th , 9th and 12th days post primary inoculation (PPI) of SRBC. The HA titre was significantly (P

Cutaneous Blastomycosis and Systemic Findings

ABSTRACT Blastomycosis is a disease that has the ability to affect nearly any organ system, most commonly causing pulmonary disease. Diagnosis of blastomycosis therefore presents a unique problem for clinicians because of its diverse array of presentations. Cutaneous disease secondary to pulmonary disease may be the initial manifestation of blastomycosis. Cutaneous disease also has variable presentations from pustules to warty verrucous or ulcerated lesions. This article will focus on the cutaneous presentation of blastomycosis, including primary inoculation and secondary disseminated types, as well as other manifestations of blastomycosis in other organ systems. Diagnostic methods and general treatment guidelines will also be discussed.

Effects of methylsulfonylmethane and neutralizing anti–IL-10 antibody supplementation during a mild Eimeria challenge infection in broiler chickens

A 28-day experiment was conducted in broilers to study the effects of feeding methylsulfonylmethane (MSM) and IL-10–neutralizing antibody from dried egg product (DEP) on the growth performance, immune responsivity, oxidative stress parameters, and gut health outcomes during a mild infection with mixed species of Eimeria. A total of 500 male Ross 308 chicks were allocated to five treatments: sham-inoculated (uninfected) chickens fed control diet (UCON), Eimeria-infected chickens fed control diet (ICON), and Eimeria-infected chickens fed control diet supplemented with 287 U/tonne of DEP (I-DEP), 0.4% MSM, or their combination (I-DEP-MSM), with 10 replicate cages of 10 birds per treatment. All infected groups received 1 mL of an oral inoculum containing Eimeria acervulina (10,000 oocysts), Eimeria maxima (5,000 oocysts), and Eimeria tenella (5,000 oocysts) on study days 7 and 14. Data were analyzed as a two-way ANOVA for all treatments including Eimeria-infected groups, in addition to a single degree of freedom contrast to compare uninfected and infected groups receiving the control diet. Mild Eimeria infection did not influence the growth performance in ICON compared with UCON at any time points. Overall (day 0–28) growth performance parameters were not influenced by either infection or dietary supplementation of MSM or DEP. However, birds in I-DEP-MSM showed improved ADG during study day 7 to 14 (i.e., 7 d after primary inoculation) indicating a beneficial effect immediately after Eimeria infection. Although MSM supplementation reduced thiobarbituric acid reactive substances (day 21 and 28), both MSM and DEP improved the total antioxidant capacity (day 21) in the plasma of infected birds. Histopathological outcomes were not influenced by treatments, and fecal oocyst output was higher in MSM- and DEP-supplemented groups than with ICON, indicating no beneficial effects. Similarly, expression of cecal inflammatory cytokines (IL-10, IL-1β, and interferon-γ) was not affected by MSM, DEP, or their combination. Overall, the current results suggest that both MSM and DEP supplementation may benefit birds during a mild Eimeria infection as indicated by improvements in ADG and oxidative stress outcomes.

Systemicacquired resistance specific proteome of Arabidopsis thaliana.

A comparative proteomic study between WT and SAR-compromised rsi1/fld mutant reveals a set of proteins having possible roles in the SAR development. A partly infected plant shows enhanced resistance during subsequent infection through the development of systemic acquired resistance (SAR). Mobile signals generated at the site of primary infection travel across the plant for the activation of SAR. These mobile signals are likely to cause changes in the expression of a set of proteins in the distal tissue, which contributes to the SAR development. However, SAR-specific proteome is not revealed for any plant. The reduced systemic immunity 1 (rsi1)/(allelic to flowering locus D; fld) mutant of Arabidopsis is compromised for SAR but shows normal local resistance. Here we report the SAR-specific proteome of Arabidopsis by comparing differentially abundant proteins (DAPs) between WT and fld mutant. Plants were either mock-treated or SAR-induced by primary pathogen inoculation. For proteomic analysis, samples were collected from the systemic tissues before and after the secondary inoculation. Protein identification was carried out by using two-dimensional gel electrophoresis (2-DE) followed by tandem mass spectrometry. Our work identified a total of 94 DAPs between mock and pathogen treatment in WT and fld mutant. The DAPs were categorized into different functional groups along with their subcellular localization. The majority of DAPs are involved in metabolic processes and stress response. Among the subcellular compartments, plastids contained the highest number of DAPs, suggesting the importance of plastidic proteins in SAR activation. The findings of this study would provide resources to engineer efficient SAR activation traits in Arabidopsis and other plants.

Mechanism of ultrasound-induced microstructure modification in Al–Zr alloys

This work probes the effect of ultrasonic melt treatment (UST) on primary intermetallic particles and α-Al using pure Al and Al–Zr alloys (Al–0.3Zr–0.1Ti, Al–0.5Zr, Al–0.5Zr–0.5Mg–0.9Si) solidified at various cooling rates of 0.2–70 K s−1. The application of UST is shown to decrease the size and increase the number density and volume fraction of D023-structured primary Al3Zr and Al3(Zr,Ti) particles with a high nucleation potency for α-Al formation. High-resolution transmission electron microscopy analysis reveals that the cavitation-induced wetting and dispersion of γ-Al2O3 inoculant particles contribute to the refinement of primary intermetallic particles. The grain size of Al–Zr alloys increases with increasing cooling rate because of the concomitantly reduced formation of primary intermetallic inoculant particles. The UST-induced refinement of primary Al3Zr particles and the increased number density lead to grain refinement in Ti-free Al–Zr alloys. For the Ti-containing Al–Zr alloy, UST-induced grain refinement is achieved only at a very low cooling rate of 0.2 K s−1, possibly because of the side effects of UST on the growth-restricting influence of Ti. The fast cooling–induced solidification enables significant age-hardening by L12-Al3Zr nanoprecipitation (~5 nm) and is not affected by UST. Thus, this study shows that UST can be used to reduce the degree of grain coarsening of age-hardenable Al–Zr alloys at high cooling rates.

Salmonella enterica serovar Enteritidis ghosts displaying a surface FliC adjuvant elicit a robust immune response and effective protection against virulent challenge

Salmonella Enteritidis (SE) is one of the most common culprits of foodborne disease in humans due to its horizontal transmission from infected animals to humans. The development of a safe vaccine against Salmonella would be important for both farm animals and humans concerning disease containment. The SE ghosts carrying FliC were genetically constructed using a special ghost plasmid pJHL184 that co-expressed FliC and the phage lysis gene E. These SE ghosts were characterized by ghost generation efficacy by increasing the culture temperature to “42 °C” in the absence of L-arabinose. This temperature change led to an ghost generation with almost complete lysis of the SE host strain in 48 hs. The expression of FliC was confirmed by Western blot analysis. Also, indirect ELISA was used to prove FliC specific antibody generation in immunized mice. The parenteral adjuvant effect of the FliC antigen was demonstrated by immunizing mice with pJHL184::flC, pJHL184 alone, or PBS alone. The mice were intramuscularly immunized at six weeks of age (n = 8) and boosted after three weeks of primary inoculation. A total of 32 mice were equally divided into four groups. Each group was treated with pJHL-ghosts alone, ghost surface displaying FliC adjuvant, and compared to the PBS and naïve control groups. The immunized mice demonstrated greater IgG and IgA antibody responses than did the PBS control group. Furthermore, the addition of the ghosts to the FliC led to a significant increase in both the humoral and cell-mediated immune responses compared to those in the ghost alone group. Besides, the in vitro antigen uptake and presentation studies revealed efficient antigen presentation on the mouse macrophage cell surfaces. This finding further corroborated the potential efficacy of immune stimulation by SE ghosts. After the virulent challenge, we observed a significant reduction in the bacterial load in the spleen and liver tissues in SE ghosts surface, displaying FliC adjuvant. Our results demonstrate a safe and effective strategy to prevent salmonellosis. They also suggest that the surface expression of flagellin (FliC) significantly enhances antigen-specific humoral and cell-mediated immune responses. This FliC expression can also enhance the protective efficacy of the bacterial ghosts-based vaccine against virulent challenge.

Rabbit VX2 lung tumor models can form early nodal metastases

BackgroundThe rabbit squamous cell cancer line, VX2, has been used to generate various tumor models in rabbits. It is notable for its ability to generate nodal metastases. However, the timing and extent of nodal metastases vary by primary inoculation site and methodology. The development of metastases specifically in lung cancer models has not been well-described. We sought to characterize the generation of nodal metastases in rabbit transbronchial VX2 lung tumor models.MethodsRabbit VX2 lung tumor models were created in the right lung via transbronchial injection and serially imaged by computed tomography. Rabbits (n = 15) were sacrificed from between 5 and 24 days post-inoculation for collection of the ipsilateral and contralateral paratracheal lymph nodes. These underwent histopathological evaluation for metastases using hematoxylin and eosin as well as cytokeratin AE1/AE3 immunohistochemical staining.ResultsNodal metastases were detectable as early as 1 week after inoculation but were more prevalent with longer inoculation; all rabbits at > 2 weeks post-inoculation had nodal metastases. Contralateral metastases were in general seen later than ipsilateral metastases. Lymph node volume did not predict the likelihood of nodal metastases (p = 0.4 and p = 0.07 for ipsilateral and contralateral nodal metastases, respectively), but primary tumor volume was significantly associated with the likelihood of nodal metastases (p = 0.001 and p = 0.005 for ipsilateral and contralateral nodal metastases, respectively). Ipsilateral metastases were detectable at a tumor diameter of 1 cm; contralateral metastases were more variable but in general required a tumor diameter of 2 cm.ConclusionsRabbit transbronchial VX2 lung tumor models generate nodal metastases relatively early after inoculation. These results suggest such models may be valuable tools in the investigation of novel therapeutic modalities relevant for the treatment of both early-stage and locally advanced lung cancer.

A FLORIST'S OCCUPATIONAL EXPOSURE

SESSION TITLE: Chest Infections: Find the Fungus SESSION TYPE: Fellow Case Reports PRESENTED ON: 10/21/2019 3:15 PM - 4:15 PM INTRODUCTION: Pulmonary infections have been reported in up to 79% of patients with blastomycosis. Often known as a “great pretender” given the large variability in presentation, pulmonary blastomycosis is a diagnostic dilemma even in endemic areas. Here we report a case of acute respiratory distress syndrome (ARDS) requiring extracorporeal membrane oxygenation (ECMO) in an immunosuppressed patient working as a florist with no travel history to an endemic area. CASE PRESENTATION: 54 year old male from central New York with history of autoimmune hepatitis on azathioprine and corticosteroids, presented with worsening cough, dyspnea and fever. He denied any sick contacts or travel history and worked as a florist. He failed outpatient pneumonia treatment with azithromycin and then doxycycline. His symptoms continued to progress, and was admitted to the ICU with acute hypoxic respiratory failure, requiring mechanical ventilation. Chest x-ray revealed patchy bilateral airspace disease and he was started on broad spectrum antibiotics and voriconazole. CT Chest showed extensive consolidation in right lower and middle lobe, left lower lobe and lingula, with mediastinal lymphadenopathy. He progressed to ARDS with worsening respiratory status despite maximal ventilator support and paralytics, and was emergently placed on ECMO. A bronchoscopy revealed thick copious mucous plugs and both bronchial washings and lavage were sent for culture. Initial blood cultures and bronchoscopy cultures returned negative for pathogens, however 10 days after bronchoscopy, lavage culture grew mold, later identified as Blastomyces dermatiditis. He was started on amphotericin B, and later changed to itraconazole. After initiation of an anti-fungal the patient clinically improved and was decannulated from ECMO, with eventual discharge to rehab. DISCUSSION: The diagnosis of pulmonary blastomycosis can range from an asymptomatic infection to pneumonia or to ARDS. The lung is often the primary inoculation site after exposure to disrupted soil. However this diagnosis requires a high degree of clinical suspicion even in endemic sites or those with a travel history to such areas. This case highlights a diagnostic dilemma in an immunocompromised patient, who presented with fulminant respiratory failure due to blastomycosis, with no travel history to an endemic area. Here we see a case of an occupational exposure to soil given his profession as a florist as his likely source, with his immunocompromised state leading to devastating disease. This emphasizes the importance of identifying occupational exposures especially when combined with those who are immunosuppressed, as early diagnosis and treatment will be lifesaving. CONCLUSIONS: ARDS due to pulmonary blastomycosis carries a high mortality rate highlighting the vital importance of early diagnosis and treatment. Reference #1: McBride JA, Gauthier GM, Klein BS. Clinical Manifestations and Treatment of Blastomycosis. Clin Chest Med. 2017;38(3):435-449. Reference #2: Moore NM, Proia LA. Severe acute respiratory distress syndrome in a liver transplant patient. Med Mycol Case Rep. 2018;21:1-3. Published 2018 Mar 6. https://doi.org/10.1016/j.mmcr.2018.03.005 Reference #3: Salzer H, J, F, Burchard G, Cornely O, A, Lange C, Rolling T, Schmiedel S, Libman M, Capone D, Le T, Dalcolmo M, P, Heyckendorf J: Diagnosis and Management of Systemic Endemic Mycoses Causing Pulmonary Disease. Respiration 2018;96:283-301. https://doi.org/10.1159/000489501 DISCLOSURES: No relevant relationships by Hassan Al Khalisy, source=Web Response No relevant relationships by Ioana Amzuta, source=Web Response No relevant relationships by Ritu Modi, source=Web Response

Evaluation of protective and immune responses following vaccination with recombinant MIP and CPAF from Chlamydia abortus as novel vaccines for enzootic abortion of ewes

MIP and CPAF from Chlamydia have been shown to be effective in inducing immune responses important in clearing chlamydial infections. This study evaluates the protection conferred by MIP and CPAF as novel vaccines in pregnant C. abortus challenged ewes. Fifty C. abortus sero-negative sheep were randomly allocated into 5 groups of 10 according to the treatment they were to receive (1) 100 µg of MBP-MIP (2) 100 µg CPAF (3) 50 µg MBP-MIP and 50 µg CPAF (4) Tris-buffer (negative control) (5) Enzovax (positive control). Booster inoculations were administered 3 weeks after primary inoculations. Blood samples were taken pre-vaccination and weekly for 5 weeks. Five months after vaccination the ewes were mated. Pregnant ewes were then challenged on day 90 of gestation. Blood samples taken at four time-points post challenge were analysed for IFNγ levels, TNFα and IL-10 expression and anti-chlamydial antibody levels. Vaginal swabs, placental and foetal tissue and bacterial shedding were analysed using qPCR to quantify levels of C. abortus. Enzovax was 100% effective with no abortions occurring. The MIP/CPAF combined vaccine offered the greatest protection of the novel vaccines with 67% of ewes giving birth to one or more live lambs equating to a 50% vaccine efficacy rate. MIP and CPAF administered singly did not confer protection. Enzovax and MIP/CPAF vaccinated ewes had longer gestations and lambs with higher birth weights than negative control ewes. Aborting ewes shed higher numbers of C. abortus than ewes that had live lambs, all vaccinated ewes demonstrated lower levels of bacterial shedding than negative control ewes with Enzovax ewes shedding significantly fewer bacteria. Ewes that went on to abort had significantly higher levels of IFNγ and IL-10 at day 35 post challenge and significantly higher levels of anti-chlamydial antibodies at 24 h post lambing compared to ewes that had live lambs.

Protocol: an improved method to quantify activation of systemic acquired resistance (SAR)

BackgroundPlant responses triggered upon detection of an invading pathogen include the generation of a number of mobile signals that travel to distant tissues and determine an increased resistance in distal, uninfected tissues, a defense response known as systemic acquired resistance (SAR). The more direct means of measuring activation of SAR by a primary local infection is the quantification of pathogen multiplication in distal, systemic sites of secondary infection. However, while such assay provides a biologically relevant quantification of SAR, it is hampered by experimental variation, requiring many repetitions for reliable results.ResultsWe propose a modification of the SAR assay based on the Arabidopsis–Pseudomonas syringae pathosystem exploiting the knowledge of source-sink relationships (orthostichies), known to centralize SAR-competency to upper leaves in the orthostichy of a lower primary infected leaf. Although many sources of variation such as genotypes of plant and pathogen, inoculation procedure, or environmental conditions are already taken into account to improve the performance of SAR assays, a strict leaf selection based on source-sink relationships is not usually implemented. We show how enacting this latter factor considerably improves data reliability, reducing the number of experimental repetitions for results.ConclusionsDirect selection of leaves for both primary and secondary inoculation exclusively within the orthostichy of the primary infected leaf is a key element on reducing the number of experimental repetitions required for statistically relevant SAR activation results.

Effects of dietary Enterococcus faecium NCIMB 11181 supplementation on growth performance and cellular and humoral immune responses in broiler chickens

This study evaluated the effects of dietary Enterococcus faecium NCIMB 11181 on growth performance and immune response in broiler chickens. A total of 360 1-day-old Arbor Acres male birds were randomly assigned to 4 treatments that administered different dosages of E. faecium (0, 5 × 107, 1 × 108, and 2 × 108 CFU E. faecium/kg diet). The results revealed that average daily gain (ADG) changed quadratically, while feed conversion rate (FCR) increased linearly from day 22 to 35 and day 1 to 35 (P < 0.05). Supplementation of E. faecium at 5 × 107CFU/kg diet resulted in increased ADG (P < 0.05) compared with the other groups. Birds fed with 2 × 108 CFU/kg E. faecium exhibited increased peripheral blood lymphocyte proliferation in response to concanavalin A (Con A) (P < 0.05) at day 35 and enhanced skin responses following phytohemagglutinin (PHA) injection (P < 0.05) at 12h. Serum lysozyme activity at day 21 increased linearly with dietary E. faecium concentration (P < 0.05), the highest activity was observed in the 1 × 108 and the 2 × 108 CFU E. faecium groups (P < 0.01). Serum levels of proinflammatory cytokines IL-1β, IL-2, IL-6, IFN-γ, and anti-inflammatory IL-4, IL-10 changed linearly or quadratically both at the initial and final phases (P < 0.05). In addition, BSA antibody titers were significantly increased following both primary and secondary inoculation when birds were fed with 1 × 108 or 2 × 108 CFU/kg E. faecium (P < 0.05). In comparison with other groups, birds received 5 × 107 CFU E. faecium exhibited the highest levels of serum IgG (P < 0.05) at day 35. Together, our results revealed that broiler diet supplemented with 5 × 107 CFU/kg E. faecium NCIMB 11181 was appropriate in relation to growth performance under normal conditions. Upon administration with higher dosages of E. faecium NCIMB 11181, obvious immune-stimulatory effects were observed following both cell-mediated and humoral immunity.

Administration of Dendritic Cells and Anti-PD-1 Antibody Converts X-ray Irradiated Tumors Into Effective In situ Vaccines

Danger signals and release of tumor-specific antigens after exposure to ionizing radiation can convert an irradiated tumor into an in situ vaccine. However, radiation alone is not sufficient to induce an effective systemic immune response. In this study, we investigated whether a combination of x-ray irradiation with bone marrow-derived dendritic cells (BM-DCs) and anti-PD-1 antibody (αPD1-ab) administration can enhance both local tumor control and the systemic abscopal effect in murine subcutaneous tumor models. B16/BL6 melanoma and Lewis lung carcinoma cells were examined for radiosensitivity and expression of H-2kd and PD-L1 before and after irradiation. The tumor cells were implanted subcutaneously in the left thigh of C57BL/6 mice as primary tumors. BM-DCs were induced from mouse bone marrow cells using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The primary tumors were treated with 8Gy of x-ray, followed by simultaneous intratumoral injection of BM-DCs and intraperitoneal injection of αPD1-ab. To examine the abscopal effect, the same tumor cells were also inoculated in the right thigh as metastatic tumors 4days after the primary tumor inoculation, and only the primary tumors were treated with the same protocols. Invivo analyses of tumor growth and survival rates and in vitro analyses of splenic T-cell proliferation and interferon-γ release were performed. The triple-combination treatment of x-ray irradiation with BM-DC and αPD1-ab administration inhibited primary tumor growth and significantly extended survival time in association with significant increase of T-cell proliferation and interferon-γ release. In addition, this triple-combination treatment significantly inhibited the growth of metastatic tumors. The results indicated that BM-DC and αPD1-ab administration led to the conversion of irradiated tumors into effective in situ vaccines. This combination therapy can be a promising approach to develop a novel individualized therapy for patients with solid cancers.

APD1, the unique member of Arabidopsis AP2 family influences systemic acquired resistance and ethylene-jasmonic acid signaling

Arabidopsis AP2 FAMILY PROTEIN INVOLVED IN DISEASE DEFENSE (APD1) is a member of AP2/EREBP super-family that positively regulates SA biosynthesis and defense against virulent bacterial pathogens. Here we report additional roles of APD1 in plant defense and development. We show that APD1 function is required for light-mediated defense against bacterial pathogens and systemic acquired resistance (SAR). We demonstrate that APD1 function is not required for generating SAR mobile signal at the site of primary inoculation but is required at the distal end for SAR manifestation. In addition, the APD1 function is required for PTI-induced callose deposition, defense against necrotrophic pathogen Botrytis cinerea and Alternaria alternata, which are ethylene (ET) or ethylene-Jasmonate (JA) dependent responses. Development of seedling under dark and ET is partly dependent on APD1. The mutant apd1 plants are non-responsive towards exogenous ACC application regarding apical hook formation and hypocotyl shortening, however, possess WT-like ET-mediated root growth inhibition. JA-mediated root growth inhibition is also impaired in apd1 seedlings. Altogether our results suggest that APD1 impacts multiple aspects of plant growth and development.

Antiviral Activity of Fermented Ginseng Extracts against a Broad Range of Influenza Viruses.

Ginseng products used as herb nutritional supplements are orally consumed and fermented to ginsenoside compounds by the intestinal microbes. In this study, we investigated antiviral protective effects of fermented ginseng extracts against different strains of influenza viruses in genetically diverse mouse models. Intranasal coinoculation of mice with fermented ginseng extract and influenza virus improved survival rates and conferred protection against H1N1, H3N2, H5N1, and H7N9 strains, with the efficacy dependent on the dose of ginseng samples. Antiviral protection by fermented ginseng extract was observed in different genetic backgrounds of mice and in the deficient conditions of key adaptive immune components (CD4, CD8, B cell, MHCII). The mice that survived primary virus inoculation with fermented ginseng extract developed immunity against the secondary infection with homologous and heterosubtypic viruses. In vitro cell culture experiments showed moderate virus neutralizing activity by fermented ginseng extract, probably by inhibiting hemagglutination and neuraminidase activity. This study suggests that fermented ginseng extracts might provide a means to treat influenza disease regardless of virus strains.