Proventriculus

Body-weight gain, feed consumption and feed:gain ratio were determined on a pen basis for
the period between days 16 and 25. InsP₆ hydrolysis and P net absorption in the
digestive tract (y) were calculated for each pen based on the ratio of
InsP₆ or P and Ti according to the generally accepted equation:
InsP₆ hydrolysis was calculated for the crop, duodenum/jejunum, lower ileum
and caeca. It was not calculated for the proventriculus/gizzard since this segment clearly
contained particles of different sizes, which presumably were of variable retention times
and thus were not accurately represented by the marker⁽³⁰⁾. P net absorption was calculated in the duodenum/jejunum and lower
ileum.
The percentage of InsP₃, ∑InsP₄ or ∑InsP₅ in
∑InsP_3–5 was calculated for each treatment and segment to investigate the
rapidity of InsP₆ hydrolysis and the extent to which intermediary products with
different degrees of phosphorylation were formed. Because InsP_1–2 isomers were
not determined, ∑InsP_3–5 was calculated, representing the sum of identified
InsP₆ hydrolysis products.
Untransformed data are expressed as means with their standard error or the pooled
standard error of the mean. Statistical analysis was performed using the MIXED procedure
of the software package SAS for Windows (version 9.1.3; SAS Institute Inc.). Before
statistical analysis, data that showed non-normal residuals or heterogeneity of variance
were log- or square root-transformed. For data expressed as percentages the arc-sine
transformation was used. The following statistical model was chosen:
y_ij = μ + r_i + τ_j + e_ij, where
y_ij is the ith measurement in the jth
treatment, μ is the overall mean, r_i is the ith block
(random), τ_j is the effect of the jth treatment (fixed) and
e_ij is the error term. Statistical significance was evaluated by a one-way
ANOVA. Mean separation was computed using Fisher's protected least significant difference
test (P ≤ 0·05) only if the overall F test was
significant (P ≤ 0·05).

Quantitative PCR was performed in triplicates using a SYBR Green kit SensiFAST™ SYBR® No-ROX (Bioline, Sydney, Australia) with Rotorgene 6000 real-time PCR machine (Corbett Research, Sydney, Australia). The PCR reaction was performed in a volume of 10 μL containing 5 μL of 2× SensiFAST, 400 mM of each primer and 2 μL of cDNA template. After thermal cycling, amplification cycle (Cq) values for all genes were collected and imported into qBase+ version 3.0 (Biogazelle, Zwijnbeke, Belgium) software and analyzed against two optimized reference genes, HPRT1 and TBP, in the current study. Both reference genes were applied to the samples of each tissue. The qBase+ applied an arithmetic mean method to transform logarithmic Cq value to linear relative quantity using exponential function for relative quantification of genes [22 , 23 ] and the output data was exported to SPSS statistics version 22 (IBM SPSS, UK) for further analysis. The relative expression levels of the genes in respective treatment groups are expressed as means of normalized relative quantities (NRQ). Relative quantity for individual genes was scaled to the lowest average expression level of the treatment being 1.
The genes analyzed in the tissues are listed as follow: PGA5, PGC and CCK, in proventriculus; AMY2A, ATP1A1, CCK1R, CCK, CELA1, CELA2A, PNLIP, in pancreas; and APN, ASCT1, ATP1A1, B^oAT, CAT1, CAT2, CCK1R, CCK, EAAT3, b^o,+AT, GLUT1, GLUT2, LAT1, PepT1, PepT2, SI, y⁺LAT1, y⁺LAT2, and rBAT in the intestine.

An adult mouse skin affixed to an artificial feeding device maintained at 37°C was used (23 (link)). In the infectious blood meal, fleas were allowed to feed for 1 h. To determine if Ail affected colonization insects fed blood containing the Δail mutant, Y. pestis KIM6⁺, or the Δail/ail⁺ complemented strain were collected, cold immobilized, and fleas that took a blood meal were identified under a dissecting microscope by the presence of fresh blood in the proventriculus or midgut. Three days after the infectious feed a 10 fleas/strain were collected and frozen at −80°C for later determination of bacterial numbers (designated 72 h). This experiment was repeated four times on separate days. Remaining insects were fed with sterile defibrinated Swiss Webster mouse whole blood (Bioreclamation) or defibrinated whole human blood (Bioreclamation) and selected as above. Fleas were kept for another 24 h in a maintenance chamber, collected, and frozen at −80°C (designated 96 h). To evaluate infection prevalence and infection rate, fleas were thawed, individually homogenized in 100 μL of a 10% glycerol solution in BHI, plated on BHI agar, and incubated 48 h at 28°C. Fleas were designated uninfected when no colonies grew from plating 15% of flea lysate. The latter experiment was repeated four times on separate days. All human blood used in these flea experiments was shown to have bactericidal activity against Y. pestis by using the serum resistance assay protocol described above.

For the histopathological and immunohistochemical examination, tissue samples collected from the brain, trachea, lung, heart, liver, spleen, proventriculus, pancreas, duodenum, ileum, and kidney were fixed in 10% neutral buffered formalin. The tissues were then routinely processed and embedded in paraffin. For histopathology, the paraffin blocks were cut on microtome into 4 µm-thick sections, which were placed on standard glass slides and stained with haematoxylin and eosin (H&E). For immunohistochemistry, the tissue sections cut from the paraffin blocks were placed onto Superfrost glass slides (Menzel–Glaser, Braunschweig, Germany) and incubated in 37 °C overnight. Next, the sections were deparaffinized, rehydrated in descending ethanol concentrations, and subjected to endogenous peroxidase blocking using 3% solution of H₂O₂ (30%) in methanol for 10 min, followed by epitope unmasking using protease K (DAKO, Glostrup, Denmark) for 15 min at room temperature. For the detection of viral antigen, anti-influenza A nucleoprotein monoclonal antibody (HYB 340-05, Statens Serum Institute, København, Denmark) was used (dilution 1:1000, 2 h). The antibody detection was performed using a Dako REAL EnVision Detection System, Peroxidase/DAB, Rabbit/Mouse (K5007, DAKO, Glostrup, Denmark). The sections were counterstained with Mayer’s haematoxylin, dehydrated, and mounted. To confirm the specificity of the staining, sections incubated with PBS instead of the primary antibody were used. The tissues were examined under a light microscope (Axiolab, Zeiss, Jena, Germany) for evaluation of histopathological lesions in the H&E-stained sections and the detection of the immuno labelling of the viral antigen in the IHC-stained ones. For an assessment of histopathological lesions, the semiquantitative scoring system proposed by Landnann et al. [22 (link)] was applied.

Three separate experiments were conducted in birds divided into 3 groups (I–III), with 15 mallards in each group.
Group I: Twelve 6-week-old mallards were infected intraocularly and intranasally with HPAIV A/herring gull/Poland/MB082B/2016 (H5N8) at the dose of 10⁶ EID₅₀/bird in 0.1 mL. Three contact ducks were placed in the same room 24 h later.
Group II: Twelve 4-week-old mallards were infected intraocularly and intranasally with LPAIV A/mallard/Poland/141/2015 (H5N1) at the dose of 10⁶ EID₅₀/bird in 0.1 mL. Two weeks later (i.e., at 6 week of age), the ducks were infected with HPAIV A/herring gull/Poland/MB082B/2016 (H5N8) (infection route, virus dose, and volume as in group I). Three contact ducks were placed in the same room after 24 h.
Group III: Twelve 4-week-old mallards were infected intraocularly and intranasally with LPAIV A/mallard/Poland/358/2006 (H3N8) at the dose of 10⁶ EID₅₀/bird in 0.1 mL. Two weeks later (i.e., at 6 week of age), the ducks were infected with HPAIV A/herring gull/Poland/MB082B/2016 (H5N8) (infection route, virus dose, and volume as in group I). Three contact ducks were placed in the same room after 24 h.
Mallards in all groups were observed for 14 days post-infection (dpi) with LPAIV and/or HPAIV. At 2, 4, 7, 10, and 14 days after infection with HPAIV (in case of contact ducks: 1, 3, 6, 9, and 13 days post-contact (dpc)), oropharyngeal and cloacal swabs were collected from all birds, placed in viral transport medium (COPAN, Italy), and stored at −80 °C until further use (viral RNA quantification by real time RT-PCR). Additionally, 4 and 14 days after HPAIV infection, organ samples (brain, trachea, lung, heart, liver, spleen, kidney, pancreas, proventriculus, dudodenum, ileum) from 2 dead or euthanized ducks (4 dpi) or all birds euthanized after termination of the experiment (14 dpi) were collected for viral RNA quantification, histopathology, and immunohistochemistry. Blood samples were also collected at 14 dpi with LPAIV and HPAIV for the purpose of serological testing.