Biomarker Evaluation and Toxic Effects of an Acute Oral and Systemic Fumonisin Exposure of Pigs with a Special Focus on Dietary Fumonisin Esterase Supplementation

Abstract

The mycotoxin fumonisin B1 (FB1) is a frequent contaminant of feed. It causes a disruption of sphingolipid metabolism and pulmonary, hepatic, and immunological lesions in pigs depending on the exposure scenario. One sensitive biomarker for FB1 exposure is the sphinganine (Sa) to sphingosine (So) ratio in blood. The fumonisin esterase FumD, which can be used as a feed additive, converts FB1 into the much less toxic metabolite hydrolyzed FB1 (HFB1). We conducted a single-dose study with barrows allocated to one of five treatments: (1) control (feed, 0.9% NaCl intravenously iv), (2) 139 nmol FB1 or (3) HFB1/kg BW iv, (4) 3425 nmol FB1/kg BW orally (po), or (5) 3321 nmol FB1/kg BW and 240 U FumD/kg feed po. The Sa/So ratio of iv and po FB1 administered groups was significantly elevated in blood and Liquor cerebrospinalis, but no fumonisin-associated differences were reflected in other endpoints. Neither clinical lung affections nor histopathological pulmonary lesions were detected in either group, while some parameters of hematology and clinical biochemistry showed a treatment⁻time interaction. FumD application resulted in Sa/So ratios comparable to the control, indicating that the enzymatic treatment was effectively preventing the fumonisin-induced disruption of sphingolipid metabolism.

Keywords: Sa/So ratio; blood count; clinical biochemistry; clinical examination; fumonisin; fumonisin esterase; pigs; single-dose.

Figure 1

Serum concentration of (a) sphinganine (Sa), (b) sphingosine (So), and the respective calculated Sa/So ratio (c) during the course of the experimental period until 120 h post-toxin application. Data represent LSmeans (±SEM; n = 6/group) and statistical main effects were distributed as follows: (a) Sa: p_group < 0.001, p_time < 0.001, p_{group × time} < 0.001; (b) So: p_group = 0.018, p_time < 0.001, p_{group × time} = 0.166; (c) Sa/So ratio: p_group < 0.001, p_time < 0.001, p_{group × time} < 0.001. CON: control; FB1iv: 139 nmol FB₁/kg·BW⁻¹; HFB1iv: 139 nmol HFB₁/kg·BW⁻¹; FUMpo: 120 mg FB₁ + 48 mg FB₂ + 14 mg FB₃/kg diet; FumDpo: 120 mg FB₁ + 48 mg FB₂ + 14 mg FB₃/kg diet and 240 U fumonisin esterase/kg diet).

Figure 2

Concentration of (a) sphinganine-1-phosphate (Sa-1-P), (b) sphingosine-1-phosphate (So-1-P), and the respective calculated Sa-1-P/So-1-P ratio (c) in whole blood during the course of the experimental period until 120 h post-toxin application. Data represent LSmeans (±SEM; n = 6/group) and statistical main effects were distributed as follows: (a) Sa-1-P: p_group < 0.01, p_time < 0.001, p_{group × time} = 0.043; (b) So-1-P: p_group = 0.542, p_time < 0.01, p_{group × time} = 0.833; (c) Sa/So ratio: p_group < 0.001, p_time < 0.001, p_{group × time} < 0.001. CON: control; FB1iv: 139 nmol FB₁/kg·BW⁻¹; HFB1iv: 139 nmol HFB₁/kg·BW⁻¹; FUMpo: 120 mg FB₁ + 48 mg FB₂ + 14 mg FB₃/kg diet; FumDpo: 120 mg FB₁ + 48 mg FB₂ + 14 mg FB₃/kg diet and 240 U fumonisin esterase/kg diet).

Figure 3

Calculated Sa/So ratio in cerebrospinal fluid at the end of the experimental period (120 h). Data represent LSmeans (±SEM) and statistical main effect was p_group = 0.031. Columns with unlike superscripts are significantly different from each other (post hoc Student’s t-test, p < 0.05). CON: control; FB1iv: 139 nmol FB₁/kg·BW⁻¹; HFB1iv: 139 nmol HFB₁/kg·BW⁻¹; FUMpo: 120 mg FB₁ + 48 mg FB₂ + 14 mg FB₃/kg diet; FumDpo: 120 mg FB₁ + 48 mg FB₂ + 14 mg FB₃/kg diet and 240 U fumonisin esterase/kg diet). Columns with unlike superscripts (a, b) are significantly different from each other.

Figure 4

Example of HE-stained, FFPE lung tissue used for quantification of tissue and airway percentage (pig 22, FUMpo). No marked histological lesions are present.

Figure 5

Analysis of lung tissue using Halo^TM image analysis software (Indica Labs, Inc., Corrales, NM, USA, 2015), determining tissue and airway proportion of the histological specimen. Data represent LSmeans (n = 6/group) and statistical main effects were distributed as p_Airway = 0.926 and p_Tissue = 0.926. CON: control; FB1iv: 139 nmol FB₁/kg·BW⁻¹; HFB1iv: 139 nmol HFB₁/kg·BW⁻¹; FUMpo: 120 mg FB₁ + 48 mg FB₂ + 14 mg FB₃/kg diet; FumDpo: 120 mg FB₁ + 48 mg FB₂ + 14 mg FB₃/kg diet and 240 U fumonisin esterase/kg diet).

Figure 6

Development of serum (a) total cholesterol and (b) total protein in pigs allocated to one of five treatments. Data represent LSmeans (±SEM, n = 6/group) and statistical main effects were distributed as follows: (a) p_group = 0.039, p_time < 0.001, p_group×time = 0.060 (b) p_group = 0.027, p_time < 0.001, p_group×time = 0.345. Reference values for total cholesterol: 77–128 mg/dL (Kraft and Dürr [27]) and total protein: 49.6–72.4 g/L (Kixmöller [28]) for German Landrace pigs. CON: control; FB1iv: 139 nmol FB₁/kg·BW⁻¹; HFB1iv: 139 nmol HFB₁/kg·BW⁻¹; FUMpo: 120 mg FB₁ + 48 mg FB₂ + 14 mg FB₃/kg diet; FumDpo: 120 mg FB₁ + 48 mg FB₂ + 14 mg FB₃/kg diet and 240 U fumonisin esterase/kg diet).

Figure 7

Experimental Design. The trial lasted for 31 days in total. On day 25, pigs were surgically equipped with indwelling venous catheters (left and right jugular vein), followed by a recovery day. During morning feeding on day 27, animals were exposed to oral or intravenous single-dose treatment. Blood samples, indicated by red arrows in panel 3, were taken over a period of 120 h for analyses of biochemical parameters, red and white blood cell count and Sa/So analysis. Moreover, clinical examinations (indicated by stethoscope) were performed on day 27 before toxin application and then every 24 h (6 times in total).

Figure 8

Overview of histopathological analysis of HE-stained, FFPE lung specimen collected at slaughter (120 h after toxin application) in order to evaluate airway and tissue proportion. (a) Screenshot of digitized HE-stained lung specimen using image analysis software (HALO^TM, Indica Labs; Corrales, NM) (b) Selected area of quantification within one histological section (c) Example from Halo^TM Tissue Classifier module (HALO^TM, Indica Labs; Corrales, NM) with lung tissue marked in red and air-containing areas marked in green.