Average AFM₁ concentration in milk.

<p>Six vaccinated (⧫) and six unvaccinated control (▴) cows were fed 102 μg of AFB₁/day from day 1 to day 11. Data are presented as mean ± SD. Within each day, differences between vaccinated and control cows are marked (**P<0.10, *P<0.05).</p

Monitoring of anti-AFB₁ Ab titers.

<p>Mean anti-AFB₁ Ab titers of high responder cows (n = 6) following vaccination with AnAFB₁-KLH in complete (first dose) and incomplete (3 boosters at three weeks intervals) Freund's adjuvant. Two animals were removed from treated group (at 13.8 and 20.8 months post priming, respectively) for health injuries unrelated to vaccination. Partum was at an average of 9.6±0.75 months after first dose vaccine administration. Data are presented (on a logarithmic scale) as mean and SD. ▴, fourth dose of vaccine (2.1 months); , booster (13.8 months); , partum (9.6±0.75 months); ▵, AFB₁ administration (15.6 months). Titers corresponding to points with different subscripts (a, b, c, d) differ significantly as <i>post-hoc</i> evaluation by Tukey's test (P<0.05).</p

Immunization schedule.

<p>CFA: complete Freund's adjuvant; IFA: incomplete Freund's adjuvant; AlOH₃: aluminium hydroxide gel; i.m.: intramuscular; s.c.: subcutaneous; <i>N</i>: number of heifers per group. Immunization consisted of a priming dose (day 0) of 500 μg AnAFB₁-KLH or 300 μg AnAFB₁-CRM₁₉₇ with adjuvant, as indicated, followed by three doses with the same amount of conjugate with adjuvant, as indicated, at intervals of three weeks. Animals of groups 1 and 2 received a fifth vaccine dose 4 months after calving, which was at age of 27±1.1 months. A loading degree of 6.4 and 8 mol AnAFB₁:1 mol KLH was determined for batch I and II, respectively. A loading degree of 1 mol AnAFB₁:1 mol CRM₁₉₇ was determined.</p

Average concentration of AFM₁, total excretion of AFM₁, carry over rate, and milk production traits.

<p>The values were calculated at the steady state condition (days 5, 7, 9 and 11) in groups of cows with average daily production of 28.7±6.3 kg of milk, subjected to ingestion of 102 μg AFB₁/cow/day.</p

Distribution of high, low and non-responders among vaccinated heifers.

<p>Distribution of high, low and non-responders among vaccinated heifers.</p

Phospho-IkBα activation and TNF-α gene expression in PM stimulated with V_HCDR3.

<p>PM (3×10⁶/ml) were stimulated for 1 h in the presence or absence (NS) of wortmannin (4 nM), V_HCDR3, LPS or NC (all at 10 µg/ml). After incubation, cell lysates were subjected to Western blotting. Membranes were incubated with Abs to pIkBα and IkBα; pIkBα was normalized against IkBα. (<b>A</b>) *, <i>P</i><0.05 (treated <i>vs</i> untreated cells, n = 5); †, <i>P</i><0.05 (wortmannin-treated <i>vs</i> wortmannin-untreated cells, n = 5). For testing the expression level of TNF-α gene, PM (1×10⁶/ml) were cultured for 1, 6 and 18 h as above described. After incubation, total RNA was isolated and analyzed for mRNA expression with RT-PCR. Transcript copy numbers were determined by qPCR using cDNA as a template. Copy numbers were normalized against the copy number of the GADPH gene (<b>B</b>). *, <i>P</i><0.05 (treated <i>vs</i> untreated cells, n = 5). Error bars, s.e.m.</p

Primer sequences used for quantitative RT-PCR.

<p>Primers were designed with the help of Beacon Designer software (Bio-Rad, Hercules, CA, USA) and provided by Invitrogen.</p

TNF-α and IL-6 production by PM and PMN stimulated with human and/or mouse CDRs and mouse V_HCDR3 uptake by different cell populations.

<p>PM (<b>A</b>) or PM and PMN (<b>B</b>) (both 5×10⁶/ml) were cultured in the presence or absence (NS) of human and/or mouse CDRs, LPS, or NC (all 10 µg/ml) for 18 h. After incubation, TNF-α and IL-6 levels were evaluated in culture supernatants by specific ELISA assays. *, <i>P</i><0.05 (treated <i>vs</i> untreated cells, n = 7). DC, PM, PMN, and T cells (all 1×10⁶/ml) were incubated for 1 h in the presence or absence (NS) of b-V_HCDR3 or b-NC (both 10 µg/ml). After incubation, permeabilized cells were reacted with FITC-labelled mAb to biotin and analyzed by FACScan flow cytometry. Data are reported as the percentage of positive cells (<b>C</b>). *, <i>P</i><0.05 (b-V_HCDR3 treated <i>vs</i> untreated cells, n = 5). Error bars, s.e.m.</p

CDRs sequences.

<p>CDRs sequences.</p

TNF-α induced TLR-4 expression in PM stimulated with V_HCDR3.

<p>PM (5×10⁶/ml) were cultured for 1 h in the presence or absence (NS) of V_HCDR3, LPS or NC (all 10 µg/ml). After incubation, TNF-α level was evaluated in culture supernatants by specific ELISA assay (<b>A</b>). *, <i>P</i><0.05 (treated <i>vs</i> untreated cells, n = 7). PM (1×10⁶/ml) were cultured for 1 h with V_HCDR3, LPS or NC (all 10 µg/ml), in the presence or absence (NS) of mAb to TNF-α (0.5 µg/ml). After incubation, permeabilized cells were reacted with RPE-labelled mAb to TLR-4 and analyzed by FACScan flow cytometry. Values represent the percentage of positive cells (<b>B</b>) *, <i>P</i><0.05 (V_HCDR3 plus mAb to TNF-α treated <i>vs</i> V_HCDR3 treated cells, n = 7). PM (3×10⁶/ml) were cultured for 1 h as above described. After incubation, cell lysates were subjected to Western blotting. Membranes were incubated with Abs to TLR-4 and actin. TLR-4 production was normalized against actin (<b>C</b>) *, <i>P</i><0.05 (V_HCDR3 plus mAb to TNF-α treated <i>vs</i> V_HCDR3 treated cells, n = 5). For testing the expression level of TLR-4 gene, PM (1×10⁶/ml) were cultured for 1 h as above described. After incubation, total RNA was isolated and analyzed for mRNA expression with RT-PCR. Transcript copy numbers were determined by qPCR using cDNA as a template. Copy numbers were normalized against the copy number of the GADPH gene (<b>D</b>). *, <i>P</i><0.05 (V_HCDR3 plus mAb to TNF-α treated <i>vs</i> V_HCDR3 treated cells, n = 5). Error bars, s.e.m.</p