1b). The lungs were washed by cannulating the Adriamycin trachea and gently injecting/recovering (3×) 1·0 ml of PBS. The bronchoalveolar lavage fluid (BAL) was centrifuged at 300 g at 4°C for 5 min and the supernatants were stored at −20°C for cytokine analysis. The cell pellet was resuspended in 0·1 ml of 3% bovine serum albumin (BSA) and cells counted using a haemocytometer. The cells were then cytocentrifuged and stained with haematoxylin and eosin (H&E) for differential

counting based on cell morphology and staining patterns. The means of three independent counts of 100 cells in a randomized field were shown. Following bronchoalveolar lavage, the lungs were fixed with formalin. Serial sagittal sections of whole lung (3–4 µm Ivacaftor nmr thick) were cut and stained with Gomori trichome for light microscopy. At least 10 fields were selected randomly and examined. The severity of the inflammatory process in the lungs was scored by two pathologists who were blinded to group identity. The scale varied from 0 to 5 as follows: 0, no inflammation, 1, minimal; 2, mild; 3,

medium; 4, moderate; and 5, marked [35,36]. The EPO assay was performed as described previously [37]. Briefly, a 100-mg sample of tissue from each lung was homogenized in 1·9 ml of PBS and centrifuged at 12 000 g for 10 min. The supernatant was discarded and the erythrocytes were lysed. The samples were centrifuged, the supernatant discarded and the pellet resuspended in 1·9 ml of 0·5% hexadecyltrimethyl ammonium bromide in PBS saline. The samples were frozen in liquid nitrogen and centrifuged at 4°C at 12 000 g for 10 min. The supernatant was used for the enzymatic assay. Briefly, o-phenylenediamine (OPD) (10 mg) Carteolol HCl was dissolved in 5·5 ml distilled water, and then 1·5 ml of OPD solution was added to 8·5 ml of Tris buffer (pH 8·0), followed by addition of 7·5 µl H2O2. In a 96-well plate, 100 µl of substrate solution was added to 50 µl of each sample. After 30 min, the reaction was stopped with 50 µl of 1 M H2SO4 and the absorbance was read at 492 nm. Levels of IL-4, IL-5,

IL-10, TNF-α and IFN-γ were determined by bronchoalveolar lavage (BAL) of the different groups of mice with an enzyme-linked immunosorbent assay (ELISA) sandwich technique using commercially available kits (OptEIA; BD Bioscience, San Jose, CA, USA), according to the manufacturer’s protocol. The optical density (OD) values were read at 450 nm. The results were expressed as picograms per millilitre, compared to a standard curve. The levels of OVA-specific IgE in serum were determined by ELISA, as described previously [38,39]. Briefly, Maxisorp 96-well microtitre plates (nunc, Roskilde, Denmark) were coated with rat anti-mouse unlabelled IgE (1 : 250; Southern Biotechnology, AL, USA) in pH 9·6 carbonate-bicarbonate buffer for 12–16 h at 4°C and then blocked for 1 h at room temperature with 200 µl/well of 0·25% PBS-casein.

We also thank Margarete Focke-Tejkl for the synthesis of additional peptides and Theresa Kapral for providing blood from osteoarthritis patients. Conflict of interest: The authors declare no financial or commercial

conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“Deficiencies in many of the complement proteins and their regulatory molecules have ABT-888 in vivo been described and a variety of diseases, such as recurrent infections, systemic lupus erythematosus (SLE) and renal diseases, may be linked to deficiency in the complement system. Screening for complement defects is therefore of great importance. In this study, we present novel improved enzyme-linked immunosorbent assays for the functional assessment of the three individual pathways of the complement system. The method is applicable at high serum concentrations and we demonstrate that it minimizes both false negative as well as false positive results. In particular, for the functional mannose-binding lectin activity it

represents an improvement on the existing assays. In this respect, the present assays represent novel improved diagnostic protocols for patients with suspected immunodeficiencies related to the complement system. The complement system is an important Galeterone immune surveillance system in vertebrates, and elements of complement functions have also been demonstrated in several invertebrate species

Selleckchem CDK inhibitor [1]. The complement system in mammals is comprised of a large number of distinct plasma and cell-associated proteins. Activation of the complement system initiates a proteolytic cascade producing protein fragments that induce opsonization or direct killing of invading pathogens and altered host cells, and generates proinflammatory responses. Furthermore, complement is also an important link between the innate and adaptive immune responses [2,3]. There are three main pathways through which the complement system can be activated. These pathways, called the classical pathway (CP), the alternative pathway (AP) and the lectin pathway (LP), depend on different components of the complement system for their initiation. They all converge to generate the same central effector molecule, C3b, through the activity of C3-activating enzyme complexes, the C3-convertases [4,5]. The CP is initiated as a result of the binding of C1q to antibody–antigen complexes or to structures such as lipopolysaccharide (LPS) or C-reactive protein (CRP), and involves a complex of C1q with the serine proteases C1r and C1s [C1q–(C1s)2–(C1r)2]. Binding of the C1-complex leads to activation of C1s, which cleaves factors C4 and C2 yielding the CP C3-convertase C4bC2a.

Both pathogens have been found in atherosclerotic plaques [5, 6] and to induce atherogenic changes in animal

models [7, 8]. In several serological studies, high serum antibody levels to these major periodontal pathogens have been found to associate with subclinical, prevalent and future incidence of cardiovascular diseases (CVD). Therefore, periodontal pathogens or host response against them may contribute to the pathogenesis of CVD [9, 10]. Heat shock proteins (HSP) Selleckchem LY294002 are a group of highly conserved proteins found in eukaryotic and prokaryotic cells including both gram-positive and gram-negative microorganisms [11]. Among HSP families, hsp60 (GroEL) homologous are major HSP antigens in various bacteria.

They are antigenically cross-reactive and serologically detectable in a wide range of gram-negative bacteria and can be considered as key molecules for autoimmune reactions [12]. Cells express HSPs when they are exposed to various forms of stress, including temperature, oxidative injury and infection. check details Factors such as bacterial lipopolysaccharides, cytokines and mechanical stress can induce the expression of host protective human HSP60 (hHSP60) on endothelial cells. Owing to the homologous nature of HSPs among species, there may be a cross-reaction of the immune response to the HSPs of the pathogens with the hHSPs expressed by stressed endothelial cells Ketotifen of the host. It has been postulated that cross-reactivity of antibodies to bacterial HSP (GroEL) with hHSP60

on endothelial cells may result in endothelial dysfunction and the subsequent development of atherosclerosis which give rise to the concept of molecular mimicry [13]. Primarily, this double-blind placebo-controlled study was designed to answer the question if clarithromycin decreases recurrent cardiovascular events in patients with acute coronary syndrome (ACS) [14]. The sample was used for the secondary analyses to examine if salivary carriage of two major periodontal pathogens, A. actinomycetemcomitans and P. gingivalis, or periodontal status is associated with serum antibody levels to HSP 60 in patients with ACS who were followed up for 1 year. Patients.  The study population consisted of 141 patients entering the hospital with acute non-Q-wave infarction or unstable angina pectoris. The inclusion criteria for recruiting study patients, the symptoms at hospitalization as well as medication, CVD status and pre-existing CVD risk factors have been described in detail previously [14]. The study was primarily designed to answer the question if clarithromycin will decrease new cardiovascular events.

5 mg/kg) were asymptomatic. Both motor activity and acoustic startle response are sensitive measures for assessing MLN8237 concentration the effects of pyrethroids at doses far below those producing convulsions.

Infection challenge with C. albicans either pre- or post-exposure to deltamethrin caused increase in the CFU both in liver and spleen. The ability of a pathogen to cause infection depends on a successful invasion of the host, which, in turn, requires that the various host defence mechanisms are not working to their full potential. For the host, an infectious process is a highly complex situation to deal with, even in situations where the immune system is not disturbed by toxic chemicals or in the deficiency buy Adriamycin of essential nutrients [27–29]. Evidence on modulation of white blood cells and lymphocyte populations by deltamethrin is limited and seems to be dependent on age, sex and species of the animals [19]. Our study shows that deltamethrin has a potential

to compromise the immunity and impair host resistance to C. albicans infection in mice. In a recent report by Suwanchaichinda et al. [30], mice exposed to deltamethrin in soyabean oil by gavage at doses of 5 and 10 mg/kg showed immunosuppression and enhanced susceptibility to malaria infection (Plasmodium berghei). Deltamethrin induced thymus atrophy by interfering with the cell signalling cascades in male BALB/c mice injected i.p. with a single dose of 25 mg/kg [31]. The immunosuppressive properties

of deltamethrin can be ascribed to its ability oxyclozanide to diffuse into cells via its property of liposolubility and inhibitory effect on protein synthesis. These reports show that as a result of exposure to deltamethrin there is a risk of weakened resistance to infection challenge. CFU in liver remained high in deltamethrin treated mice. In spleen and liver, CFU were more than deltamethrin alone treated animals. This shows a high infection rate in spleen and liver. When mice were treated with deltamethrin before and after the challenge by C. albicans, it significantly suppressed the humoral immune response. Lymphocytes play a major role in immune defense against the bacterial infections [32, 33]. For instance, TH1 cells and interferon-γ are required to control the primary peak parasitemia in mice infected with Plasmodium chabaudi and this mechanisms is antibody-independent [34]. CD4 +  TH1 and TH2 cells and antibodies are required after the peak to eliminate the parasites. Depletion of natural killer cells can also cause a rapid increase in fungal or bacterial infection [35]. Studies on the insecticide sulfluramid caused suppression ranging from 70 to 89% (6–57 μmol/kg/day) in mice [36]. Decreases in IgG classes (IgG1, IgG2b, IgG3) were also reported following exposure to perfluoroctanic acid for 10 days [37]. Taken together, our data suggest that humoral immunological function may be a target for pesticides.

A potential caveat of the above results is that the CD3lo DP cells from Bcl11bdp−/− mice may not represent a pure population of immature,

unselected, DP cells, and might contain cells derived from more mature populations, possibly owing to the difficulty to resolve the mutant cell populations with the CD8, CD4, and CD3 markers. To address this issue, we analyzed the expression of several genes previously found to be induced in WT DP cells during positive selection, using transcriptome data from a published comparison of gene expression profiles of unselected DP cells (CD69− DP cells from Zap70-deficient mice) to selected, click here CD69hi cells from WT animals 41 (data accessible at H 89 in vivo NCBI GEO database accession GSE2262). Although some selection-induced genes were indeed overexpressed in the CD3lo DP cells from Bcl11bdp−/− mice (Zbtb7b, Id2, Klf2, CD53, IL7r, and Irf7), several others were expressed at similar low levels in WT and mutant cells (Itm2a, Nr4a1, Bcl2a1a, Slfn1, Mapk11, Nr4a3, Tnfrsf9,

Acvrl1, Ccr7, Ephx1, Ms4a4b, St6gal1, Tes, Nab2, and Ccl22), suggesting that the mutant CD3lo DP cells do not exhibit a general induction of the gene expression program associated with thymocyte maturation. We selected five of these genes (Ccr7, Slfn1, Ephx1, Ms4a4b, and Mapk11) for further analysis, as these genes displayed strong differences in gene expression levels between unselected and selected cells in the data from Sun et al.41 (>3 log induction), mafosfamide and were thus likely to be informative with respect to the selection/purity status of the analyzed populations. We sorted CD3loDP, CD3+DP, CD3+CD4+ SP, and CD3+CD8+ SP cells from two WT and two Bcl11bdp−/− mice (see Supporting Information Fig. 6 for

sorting gates and purity of the sorted populations) and analyzed the expression of the selected genes in these populations by RT-qPCR (Fig. 7). In WT samples, all five genes were expressed at low levels in CD3lo DP cells and strongly induced in the CD3+ DP and SP populations, thus validating previous microarray results 41. In agreement with our transcriptome data, all five genes were also expressed at very low levels in mutant CD3lo DP cells. Two genes (Ephx1 and Ms4a4b) were strongly induced in the mutant CD3+DP and SP-like populations. This observation reveals that the phenotypically more mature cells from Bcl11bdp−/− mice have retained the capacity to induce a subset of the genes normally upregulated during positive selection.

The income is generated through the sale of Karunya Lottery which is exclusively devoted for extending financial assistance to this purpose. Hence this finance is fully contributed by the public. Kerala government GSK126 cost started free karunya dialysis scheme 6 months ago which provide 2 hemodialysis per week irrespective of the residual renal function. We conducted a study to compare

the surogate markers of dialysis adequacy between patients undergoing hemodialysis in karunya dialysis scheme and in private sector. Methods: This was a cross-sectional observational study. All the 83 patients who were undergoing Hemodialysis under karunya scheme in our institution (Government medical college kerala,

India) and 100 patients undergoing hemodialysis in 3 randomly selected dialysis centers under private sector in our city were enrolled into our study. Patients informations were retrieved from from dialysis unit using a data entry form. The information retrieved included patients demographic data, etiology of ESRD, frequency of hemodialysis, types of vascular access used for hemodialysis, frequency of intravenous iron therapy and ESA and the frequency of blood transfusion. The surrogate markers for adequacy Metabolism inhibitor like Hemoglobin, ferritin, Albumin, Calcium, phosphrous PTH, lipid profile, signs of fluid overload, uraemic symptoms and biometrics

like midarm circumference, BMI and triceps skin fold thickness were compared between the 2 groups. We also looked at the quality of life based on WHO-QOL BREF questionaries in both the groups. Statistical analysis was done using SPSS version Nintedanib ic50 21.0. Results: There were no statistical difference between the two groups in all the parameters compared. Conclusion: Karunya free Dialysis Scheme is an effective way for improving the access to maintenance hemodialysis and renal care without compromising on the outcome and quality of life. Hence we suggest karunya model of dialysis to all End Stage Renal Disease (ESRD) patients in resouce poor countries. HUNG CHI-CHIH, CHANG JER-MING, TSAI JER-CHIA, CHEN HUNG-CHUN Division of Nephrology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University Introduction: Higher hemodialysis (HD) adequacy presented as Kt/V is associated with lower all-cause mortality in observational studies, though randomized HEMO study showed no superior survival of higher target Kt/V group patients (Kt/V 1.65 vs Kt/V 1.25). Some subgroups of HD patients such as Asian people or female may have benefits under higher Kt/V. Thus, we would ask whether higher Kt/V with the dose more than that in HEMO study would be associated better survival after long term follow-up.

[9] A necrotic eschar in maxillary, facial, or sino-orbital mucosal surfaces in an immunocompromised host may be an early PLX-4720 ic50 sentinel marker of invasive

mucormycosis. Pleuritic pain in a neutropenic host also may signify an angioinvasive filamentous fungus. Pleuritic pain in a neutropenic or HSCT patient receiving voriconazole prophylaxis has a high probability of being invasive mucormycosis instead of aspergillosis. Diplopia is an early manifestation of sino-orbital mucormycosis in a diabetic patient that usually signifies involvement of the extraocular muscles or their innervating nerves.[10] Hyperglycaemia in diabetic patients may produce blurring of vision, but does not produce diplopia. During sino-orbital mucormycosis, hyphae involving the ethmoid sinus breach the lamina papyracea to invade the medial rectus muscle creating dysconjugate vision. The organism may extend along the emissary veins to the ethmoid sinus to the cavernous sinus and encroach upon the critical cranial nerves involve III, IV, V (1, 2) and VI. Diplopia in a diabetic patient or other compromised host with ethmoidal sinusitis should be assessed aggressively for sino-orbital mucormycosis. Necrotic cutaneous lesions in immunocompromised

patients may also be caused by mucormycosis. The differential diagnosis includes Roscovitine cell line other angioinvasive pathogens including Aspergillus, Fusarium, Pseudallescheria, Scedosporium species. Pseudomonas aeruginosa and occasionally members of Enterobacteriaceae in the same host also cause ecthyma gangrenosum. The preponderance

of cases of cutaneous mucormycosis is associated with direct inoculation rather than haematogenous dissemination.[1] Characteristic hyphal structures are seen on biopsy 3-mercaptopyruvate sulfurtransferase and wet mount of tissue. Earlier recognition of sinus and pulmonary lesions by CT scanning is an important advance over conventional sinus and chest radiographs. Early CT findings may reveal pulmonary or sinus lesions before localising symptoms in immunocompromised patients who are at high risk for invasive sino-pulmonary mucormycosis. Among the lesions associated with angioinvasive filamentous fungi are nodules, halo signs, reverse halo signs, cavities, wedge-shaped infiltrates and pleural effusions associated with pleuritic pain.[11] Among these lesions, the reverse halo sign in the neutropenic patient has high predictive value for mucormycosis.[12] Early recognition of risk factors, clinical manifestations and diagnostic imaging findings may increase the probability of an early recognition and lead logically to a definitive diagnosis by culture and biopsy of tissue or the use of novel molecular and antigenic assays.

Goblet cell counts showed a major increase, as did eosinophils in relation to naïve controls. Paneth cells were also elevated, but did not change over the course of the experiment. The results also drew attention to the tremendous resilience of hookworms, some adult worms surviving throughout, despite highly inflamed intestines. In humans, hookworm infections are typically long-lasting, and despite much research over the last decades, there is still little evidence that a strong protective immunity to the parasite is generated, at MG-132 research buy least

at the population level (1–4). One explanation for this may be that in the current period of evolutionary history and in the context of the continuing arms’ race between parasites and their hosts, human hookworms have temporarily gained the upper hand and that consequently, for the present, their evasive mechanisms are generally more effective than the host-protective mechanisms available to human hosts to counteract infection. Data exist to indicate that hookworms manipulate host responses, down-regulating host immune capacity in their own favour (5–7). Epidemiological studies NVP-AUY922 have shown, nevertheless, that some individuals can live in endemic regions without acquiring heavy infections and it is known that there is a genetic component that governs susceptibility/resistance to infection in humans

(8–10). In contrast to the chronic infections experienced by humans, animals can resist hookworms effectively. For example, dogs show strong acquired immunity to their hookworms (11–13). Unfortunately, rodents do not have their own hookworm species (members

of the family Ancylostomatidae) that can be used to dissect the complex interactions between these haematophagous parasites and their hosts. However, some canine and human hookworms have been adapted for hamsters, and these have attracted increasing attention as model systems for exploring further the host–parasite relationships of Methane monooxygenase hookworms (13,14). The hamster-Ancylostoma ceylanicum model is one that has been particularly popular in this context in recent years (6,15). Hamsters tolerate a chronic primary infection with A. ceylanicum which can last for well over 100 days, although heavier infections are controlled slowly with worm numbers declining gradually over many weeks (14,16), rather than rapidly over just a few days as for example, in the case of Trichinella spiralis in mice (17). Low-intensity primary hookworm infections show little change in worm burdens for even longer (16). Hookworms are extremely resilient and can tolerate and survive in highly inflamed intestinal tissues (5). During primary infections mast and goblet cell numbers are elevated, as are eosinophil numbers in the hamster mucosa (18) and hookworm-specific antibodies are produced both in the serum and the intestine (6,15,19).

Cells were stained with TMRE (Sigma-Aldrich, St Louis, MO, USA) in PBS to a final concentration of 125 nM, and incubated for 30 min at 37°C with 5% CO2 to assess mitochondrial membrane potential (ΔΨm). Total mitochondrial mass and membrane potential were also determined using mitotracker green and red dyes (Invitrogen), respectively, according to manufacturers’ instructions. For in vitro culture experiments, CD8+ T cells were purified >90% by magnetic-activated cell sorting (MACS) using anti-CD8α microbeads learn more and LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany) following manufacturer’s instructions. For microarray and Western blot analysis,

CD8+ T cells were purified >98% using the Easysep PE selection kit (StemCell Technologies) using PE-CD8α (eBioscience). Primary naïve CD8+ T cells were cultured in 24-well plates at 1×106 cells/mL at 37°C with 5% CO2 in RPMI-1640 medium (Sigma-Aldrich) supplemented with glutamine, 2-mercaptoethanol and antibiotics (all Sigma-Aldrich). Where used, IL-7 (Peprotech, Rocky Hill, NJ, USA) was supplemented at 50 ng/mL. CD8+ T cells were sorted, selleck chemicals llc and total RNA was prepared using the RNEasy mini kit (Qiagen). RNA was quality and quantity controlled for degradation on a BioAnalyzer

2100 (Agilent). Since the starting quantity of RNA for each sample did not exceed  μg, two cycle amplification was performed, as recommended by the manufacturer (Affymetrix). The GC-RMA (Robust Multiarray Analysis) algorithm was applied to the probe level data (CEL files). Quality control and data processing was performed Sodium butyrate at the Bloomsbury Centre for Bioinformatics, University College London, using the limma,

gcrma, simpleaffy, annotate, annaffy and affycoretools R packages in Bioconductor. Multiple testing correction was applied for the data using the Benjamini and Hochberg False Discovery Rate. Annotation of each probe set was derived using the NetAffx site (Affymetrix). Microarray data were deposited in ArrayExpress (accession number E-TABM-991). Cell pellets containing 1×106 cells were lysed at 4°C in 1 mL 1% NP40 lysis buffer. Protein lysates were run on 12% SDS-PAGE acrylamide gels and protein content analysed on nitrocellulose membrane with the following antibodies: Mcl1 (Rockland), Bcl2, BclXL, Bok, Bax, total Bad, Bim, Bid, Bak, Puma, pBad (Ser112) (all from Cell Signaling Technology), and Actin (Santa Cruz). Densitometry calculations of proteins were calculated in the ImageJ v1.43 (NIH, Public Domain). The authors thank Biological Services for animal husbandry and technical support, Hugh Brady for providing Bad transgenic mice. This work was supported by the Medical Research Council UK under programme code U117573801. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset.

Susceptibility for antimicrobial agents

was tested by the broth microdilution method using Dry Plate Eiken (Eiken Chemical, Tokyo, Japan) according to the Clinical and Laboratory Standards Institute-approved procedures. The following 17 antimicrobial see more agents were tested: ampicillin, ceftiofur sodium, streptomycin, gentamicin, kanamycin, tetracycline, bicozamycin, chloramphenicol, enrofloxacin, orbifloxacin, norfloxacin, danofloxacin, ofloxacin, sulfonamide + trimethoprim, colistin base, sulfadimethoxine, and nalidixic acid. The quinolone resistance-determining regions of the gyrA, gyrB and parC genes were amplified as described previously (11, 40). Lethality tests were performed using 5-week-old SPF white leghorn chickens. Sixty chickens were allotted to six groups (10 birds per group). The chickens in three of the groups were injected i.v. with 1.6 × 109 CFU, 1.6 × 108 CFU or 1.6 × 107 CFU of the mutant strain (AESN1331); the chickens in the three other group were injected i.v. with 2.0 × 109 CFU, 2.0 × 108 CFU, or 2.0 × 107 CFU of the parent strain (J29). The volumes of all injections were 0.5 mL. The chickens were observed for the subsequent 14 days, and the LD50 calculated by the method of Reed and Muench (41). In vivo colonization by the mutant was assessed using 4-day-old SPF white leghorn chickens.

Forty-eight chickens were allotted to two equal groups. www.selleckchem.com/products/Liproxstatin-1.html One group was given 109 CFU/bird of AESN1331, and the other group 109 CFU/bird of J29. All doses were

administered by fine spray at volumes of 0.3 mL per chicken. On day 1 and then 1, 2, 3, 4, 5, and 6 weeks post inoculation, three birds per group per time point were killed and necropsied. For bacteriological assessment using DHL agar plates (Eiken Chemical) supplemented with nalidixic acid (0.025 mg/mL), the hearts, livers, spleens, lungs, cecums, and bursas of Fabricius were aseptically recovered, and the nasal and orbital cavities, tracheas, air sacs, and articular cavities swabbed with sterilized CYTH4 cotton. An additional three inoculated chickens per group were killed at 7 days post-inoculation, and the hearts, livers, spleens, bursas of Fabricius, and tracheas of each bird submitted for histopathological examination using standard techniques. Forty SPF white leghorn 4-day-old chickens were allotted to four equal groups for inoculation as follows: fine spray, coarse spray, eye drop, or no treatment (unimmunized). In the three treated groups, bacteria (3 × 107 CFU of AESN1331 per bird) were administered by the indicated route twice, at 4 and 32 days of age. Fine spray, delivered as droplets of < 100 μm in diameter, was administered at 0.3 mL/bird/dose using a New-con 607 (Thomas Industries, Louisville, KY, USA). Coarse spray, delivered as droplets of < 100 μm in diameter, was administered at 0.3 mL/bird/dose using a Pana-Spray (Panasonic, Osaka, Japan).