Zinc status and indicators of intestinal health in enterotoxigenic Escherichia coli F18 challenged newly weaned pigs fed diets with different levels of zinc

Abstract

This study investigated the impact of an enterotoxigenic Escherichia coli (ETEC) F18 challenge on newly weaned pigs when fed one of three Zn levels (150, 1,400, or 2,500 ppm) on performance, Zn status, ETEC shedding, and diarrhea. The ETEC challenge was hypothesized to have a more pronounced negative impact on pigs fed a diet containing 150 ppm Zn compared to 1,400 or 2,500 ppm Zn for 14 d after weaning. The study included 72 ETEC F18 susceptible pigs weaned at ~28 d of age (d 0 of the study). The pigs were distributed according to initial weight and litter to one of the three dietary Zn levels. Half of the pigs were challenged with ETEC on d 1 and 2. The challenge reduced (P ≤ 0.03) feed intake and average daily gain (ADG) during d 3 to 5. Challenged pigs fed 150 ppm Zn had lower (P = 0.01) ADG during d 5 to 7 compared to those fed 1,400 or 2,500 ppm Zn, whereas control pigs’ ADG were not affected by dietary Zn content. Challenged pigs fed 150 ppm Zn also showed lower (P < 0.01) fecal dry matter (DM) on d 5 compared to control pigs fed 150 ppm Zn and challenged pigs fed 1,400 or 2,500 ppm Zn. Challenge increased (P < 0.01) ETEC shedding in all groups, but challenged pigs fed 150 ppm Zn showed higher (P ≤ 0.05) fecal shedding of ETEC and toxins than when fed 1,400 or 2,500 ppm. On d 3, C-reactive protein concentration in plasma was lower (P < 0.03) for pigs fed 1,400 and 2,500 compared to 150 ppm Zn. Plasma haptoglobin and pig major acute phase protein were unaffected by dietary Zn content. On d 0, the serum Zn concentration was 586 ± 36.6 µg/L, which pigs fed 150 ppm Zn maintained throughout the study. The serum Zn concentration increased (P ≤ 0.07) in pigs fed 1,400 or 2,500 ppm Zn. The challenge decreased (P < 0.01) the serum Zn concentration in pigs fed 2,500 ppm Zn. On d 5 and 7, serum Zn concentration was similar for challenged pigs fed 1,400 and 2,500 ppm Zn, while control pigs fed 2,500 ppm Zn had higher (P < 0.01) serum Zn concentration than 1,400 ppm Zn. On d 7, serum Zn concentration tended (P = 0.08) to be lower for pigs with diarrhea (fecal DM ≤ 18%). In summary, these results indicate that newly weaned pigs fed 150 ppm Zn are more susceptible to ETEC F18 colonization and its adverse consequences such as diarrhea and reduced growth, even though challenge did not increase acute phase proteins.

The negative impact of enterotoxigenic Escherichia coli F18 on the growth performance and prevalence of diarrhea in newly weaned pigs is aggravated by a low dietary zinc concentration (150 ppm). The challenge with enterotoxigenic E. coli F18 reduced the serum zinc concentration for pigs fed a high dietary zinc concentration (2,500 ppm).

Introduction

Weaning is a stressful phase for pigs as they encounter multiple changes, such as separation from their dam (loss of maternal immunity), transition to plant-based solid feed, and acclimation to a new environment alongside unfamiliar pen-mates (de Groot et al., 2001; Moeser et al., 2007). Also, the metabolizable energy intake can be reduced by up to 80% immediately after weaning, taking ~2 wk to return to the preweaning level (Le Dividich and Sève, 2000). The low feed intake after weaning increases the paracellular permeability (Spreeuwenberg et al., 2001), which again increases the possibility for translocation of luminal bacteria and microbial products. This is critical, as the immune system is not fully developed at the typical weaning age (d 21 to 28 of age; Moeser et al., 2017).

Enterotoxigenic Escherichia coli (ETEC) is one of the most common pathogens newly weaned pigs encounter (Fairbrother et al., 2005), which can alter the intestinal barrier function, leading to diarrhea, reduced growth performance, and increased mortality in the period after weaning (Gao et al., 2013; Ngendahayo Mukiza and Dubreuil, 2013; Lei and Kim, 2020). Both ETEC F4 and F18 induce diarrhea in pigs weaned around d 28 of age, as F4 receptors are expressed from birth, and F18 receptors are found in 10 d old pigs (Nagy et al., 1992; Coddens et al., 2007). Danish pigs are generally more likely to be susceptible to ETEC F18 than F4, as only few sows are homozygote for the F4 receptor due to the Danish breeding program (The National Committee for Pig Production, 2004).

Zinc deficiency can increase the susceptibility to gastrointestinal infections as it may impair the intestinal barrier function (Wang et al., 2022). Currently, the EU allows a maximum dietary Zn content of 150 ppm in weaned pigs diets. This only potentially meets the NRC requirement of 46.8 mg/d (7 to 11 kg body weight; BW; NRC, 2012; EU, 2016) since the feed intake during the first week after weaning is often below 200 g/d and therefore not enough to ensure adequate Zn intake (Bruininx et al., 2001; NRC, 2012).

Serum Zn concentration is a continuous biomarker for Zn status, and concentration below 650 to 700 µg/L is considered deficient in pigs as well as in humans (Puls, 1990; Hotz et al., 2003; Hess et al., 2007). At weaning (21 to 28 d of age), pigs typically have a serum Zn concentration of ~600 to 800 µg/L, which may be just within the presumable sufficiency level (Jensen-Waern et al., 1998; Josa, 2016; Hansen et al., 2022b; Nielsen et al., 2022). Studies by Hansen et al. (2022b) and Nielsen et al. (2022) showed a 20% decline in serum Zn concentration within the first 2 wk after weaning when pigs were fed ~150 ppm dietary Zn. Thus, 150 ppm dietary Zn potentially increases the risk of Zn deficiency, which could result in a lower resistance to infections.

Our previous study established the optimal dietary Zn content during the first 2 wk after weaning to 1,400 ppm based on feed intake, growth, and serum Zn concentration (Hansen et al., 2022b). However, the study was conducted in a research facility with a higher hygiene level and thereby probably a lower pathogen load compared to commercial pig farms. Information is lacking on the impact of dietary Zn content and E. coli infection on Zn status and intestinal health in weaned pigs. Therefore, this study’s objective was to investigate the effect of experimentally challenging newly weaned pigs with ETEC F18 on growth performance, diarrhea-related parameters, and serum Zn concentration while feeding a diet containing 150, 1,400, or 2,500 ppm Zn. It was hypothesized that the ETEC challenge would have a greater negative impact on pigs fed 150 ppm Zn compared to 1,400 or 2,500 ppm and that the challenge would lead to a less effective response of acute phase proteins in pigs fed 150 ppm dietary Zn.

Materials and Methods

The animal experimental procedures were carried out in accordance with the Danish Ministry of Justice, Law no. 474/15.05.2014 concerning animal experiments and care and license issued by the Danish Animal Experiments Inspectorate, Ministry of Food, Agriculture and Fisheries, the Danish Veterinary and Food Administration.

Experimental animals and housing

A total of 72 crossbred pigs (36 males and 36 females; initial BW 6.71 ± 0.28 kg; [Danish Landrace × Yorkshire] × Duroc) from 12 sows (6 pigs/sow) were weaned at ~28 d of age (d 0) and were obtained from a commercial farm in three batches of 24 pigs each. The 12 sows, from which the pigs were obtained, were confirmed homozygote carriers of the dominant gene (FUT1^GG) encoding ETEC F18 fimbriae receptors by VHL genetics (Wageningen, The Netherlands); thus, pigs were genetically susceptible to ETEC F18 (Meijerink et al., 1997). The sex of the pigs was not considered in the experimental design.

Upon arrival at the experimental facilities, the 24 pigs in each batch were housed in groups of two pigs (from two different sows) per pen (1.5 × 2.4 m) in two rooms (12 pigs in six pens per room). One-third of the pen floor area was slatted, and the concrete part of the floor had a cover and floor heating. An empty pen between the pens with the pigs ensured that pigs from different pens had no physical contact. The rooms were ventilated with neutral pressure and linked to the temperature sensors. The initial temperature was set to 25 °C and adjusted weekly to reach 23 °C at the end of the study. The pigs were provided a 12-h light/dark cycle.

Experimental design, diets, and feeding

The pigs were fed ad libitum one of three dietary Zn levels (150, 1,400, and 2,500 ppm) during the 14-d experimental period, and the diets were given to pigs in both rooms (2 pens/diet/room). The basal diet, to which the various Zn levels were added, had the same ingredient composition as used by Hansen et al. (2022b). High-purity ZnO (80% Zn, VetZink, Vepidan ApS, Løgstør, Denmark) was used as Zn supplementation and the diets were treated similarly as in Hansen et al. (2022b), except that cereals in the present study were milled through a 3 mm screen before being mixed with the remaining ingredients.

On d 1 and 2, in each batch, 12 pigs from one room were orally inoculated with 5 mL NaCl (0.9%) containing ~10⁹ CFU ETEC F18/ml (5 × 10⁹ CFU/pig/d). The pigs in the other room received 5 mL of NaCl (0.9%). The E. coli or saline solution was administrated through a polyethylene tube connected to a syringe placed in the mouth of the pig. The ETEC strain (O138 F18-ETEC 9910297-2STM) used expressed F18 fimbriae, heat-stable enterotoxin b (STb); heat-labile enterotoxin (LT); enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1), and Shiga toxin type 2e (Stx2e), and was isolated from the intestinal content of a pig with post-weaning diarrhea. The strain was provided by the Danish Veterinary Institute (Copenhagen, Denmark). The inoculum was prepared as described by Jerez-Bogota et al. (2023).

The ETEC challenge of pigs in one room per batch created six treatments with three dietary Zn levels and two challenge levels in a 3 × 2 factorial arrangement.

Experimental procedure

At the beginning of each batch and at the end of the study, representative samples of the experimental diets were obtained. At the end of the study, samples from each diet were pooled, and a representative sample was obtained and used for chemical analysis.

The average daily gain (ADG) was calculated by weighing the individual pigs on d 0 (at arrival), 3, 5, 7, and 14 (end of the experiment). Pigs were euthanized if they lost more than 15% of their initial BW or if they showed signs of severe illness. The average daily feed intake (ADFI) was calculated on pen level (2 pigs/pen) by weighing the feed residues on d 3, 5, 7, and 14, and calculated as the average ADFI per pig.

The feces consistency in the pens was assessed daily based on a visual four-category scale (1 = firm and shaped; 2 = soft and shaped; 3 = loose; 4 = watery) with fecal scores 3 and 4 classified as diarrhea (Pedersen and Toft, 2011). Moreover, fecal samples were obtained from each pig directly from the rectum at d 0, 3, 5, 7, and 14 to determine dry matter (DM) content for a more objective measurement of diarrhea (stored at −20 °C) and for quantitative polymerase chain reaction (qPCR; stored at −80 °C).

Blood samples were obtained from all pigs by puncturing the jugular vein. Serum samples for Zn analysis were collected in vacutainers specifically for mineral analysis (Becton Dickinson AS, Kongens Lyngby, Denmark) on d 0, 5, 7, and 14. Samples for acute phase proteins were collected in EDTA vacutainers (to obtain plasma) on d 0, 3, 5, 7, and 14. The serum samples stood at room temperature for 1 h before being centrifuged. Both samples were centrifuged at 1,300 g and 4 °C for 10 min. and stored in polyethylene tubes at −20 °C until analyzed.

Sample analyses

Before mineral analysis, the feed and serum samples were digested with concentrated HNO₃ (67% to 69 %), followed by destruction using a microwave system (Ultra wave, single reaction chamber, Milestone, Shelton, USA). The Zn and Cu contents were measured on an iCAP TQ ICP-MS (Inductively Coupled Plasma-Mass Spectrometer) as described in detail by Hansen et al. (2022b). Two isotopes of Zn and Cu were measured: ⁶⁶Zn and ⁶³Cu isotope as quantifier and ⁶⁴Zn and ⁶⁵Cu isotope as qualifier. The Zn and Cu content in the feed was analyzed four times, while the Zn and Cu content in the serum was analyzed once.

Haptoglobin was analyzed by spectrophotometry using the PHASE Haptoglobin Assay Kit (TP801; Tridelta Developments Ltd., Co. Kildare, Ireland). C-reactive protein (CRP) and pig major acute protein (Pig-MAP) were determined by particle-enhanced immune turbidimetry, using the Turbovet pig CRP and Turbovet pig-MAP, respectively (Acuvet Biotech, Zaragoza, Spain). The three acute phase proteins were analyzed using an auto-analyzer (ADVIA 1800 ® Chemistry System, Siemens Medical Solutions, Tarrytown, NY 10591, USA).

The fecal DM content was determined by freeze-drying to constant weight; values lower than or equal to 18% were considered diarrhea (Pedersen et al., 2011). Quantitative polymerase chain reaction (qPCR) was used for quantification of the gene encoding the F18 fimbriae (fedA gene), LT2 toxin (eltB gene), and STb toxin (estB gene) in fecal samples following the procedure described by Jerez-Bogota et al. (2023).

Statistical analyses

Data were statistically analyzed using the RStudio version 1.4 (R Studio Team, 2021) according to a randomized complete block design in a 3 × 2 factorial arrangement with dietary Zn content (150, 1,400, and 2,500 ppm) and challenge (control vs. ETEC challenge) as factors. The experimental unit for the performance parameters (e.g., ADG, ADFI, and average daily Zn intake; ADZnI) was the pen, while the experimental unit for the blood and feces parameters (e.g., serum Zn, acute phase proteins, and fecal ETEC shedding) was the pig, whereas batch was the block. The normality of the data were verified by Q-Q plots and residual plots. Differences were considered significant at P ≤ 0.05, while P-values between > 0.05 and ≤ 0.10 were considered statistical tendencies. If the statistical analysis showed an effect (P ≤ 0.10) on dietary Zn content, a multiple comparison analysis was performed using Turkey adjustment.

ADFI, ADG, and ADZnI were analyzed using a linear mixed model with dietary Zn concentration, challenge, and their interaction as fixed effects, and pen, block, and litter as random effects. The model for ADG also included the individual BW on d 0 and sex as covariates, while the model for ADFI and ADZnI included the average BW on d 0 of the pen.

Fecal DM, serum Zn concentration, plasma concentration of acute phase proteins, and E. coli shedding were analyzed using linear mixed models with dietary Zn concentration, challenge, day, and their interactions as fixed effects, and with pig, block, and litter as random effects, and individual BW on d 0 and sex as covariates.

The correlations between serum Zn concentration and fecal DM greater or less than 18% were analyzed using linear mixed models with fecal DM as a fixed effect and block and litter as random effects. Fecal DM >18% was classified as “no diarrhea” and a fecal DM ≤18% was classified as “diarrhea.”

The correlations between ADG and E. coli shedding were analyzed using linear mixed models with shedding as a fixed effect and block and litter as random effects. The serum Zn concentrations on d 5, 7, and 14 were tested for correlation with the ADZnI during d 3 to 5, 5 to 7, and 7 to 14 analyzed using linear mixed models with shedding as a fixed effect and block and litter as random effects.

The acute phase proteins were cubic root transformed and fecal E. coli and toxins copy numbers were log-transformed to obtain the normality of the data.

Fecal scores at the pen level were analyzed by a generalized linear model with the fecal score as a binomial variable and the average BW on d 0 as a covariate. Pen fecal scores 1 and 2 were combined to 0 (no diarrhea) and fecal scores 3 and 4 were combined to 1 (diarrhea). Obtained values were square means of log odds and transformed into probabilities of transformed log odds.

Results

Animals and diets

One ETEC-challenged pig fed 150 ppm dietary Zn was euthanized on d 8 due to a loss of 15% of its initial BW, and one control pig fed 2,500 ppm dietary Zn was euthanized on d 13 due to acute paralysis.

The analyzed total Zn contents in the diets were on average 144, 1,402, and 2,589 ppm, thus it differed by 0.1% to 4% from the intended concentrations. The Cu content was similar in the three diets, and the diets contained on average 146 ± 6.6 ppm Cu.

Performance

There was a tendency (P = 0.06) for an interaction between dietary Zn content and challenge on the ADFI during d 0 to 3, with a tendency (P = 0.08) for pigs fed 2,500 ppm Zn to have a greater ADFI when challenged. However, no interaction was observed in the period after d 3 (Table 1). The ADFI during d 3 to 5 was reduced (P = 0.03) by the challenge, and there was a tendency (P = 0.08) for an effect of dietary Zn content. A dietary Zn content of 1,400 ppm increased (P = 0.01) ADFI during d 5 to 7 compared with 150 ppm, while pigs fed 2,500 ppm dietary Zn had similar ADFI as pigs fed both 150 and 1,400 ppm dietary Zn. The ADFI during d 0 to 7, 7 to 14, and 0 to 14 was not affected by dietary Zn content or challenge. Generally, the ADZnI increased (P < 0.01) with increasing Zn content in the diet, whereas there was no effect of challenge (Table 1).

Table 1.

Effect of dietary zinc (Zn) concentration (ppm) and challenge (C) on average daily feed intake, average daily gain, and average daily zinc intake (n = 12)¹

Zinc, ppm	Control			Challenge			SEM	P-values
	150	1,400	2,500	150	1,400	2,500		Zn	C	Zn × C
Average daily feed intake, g/d
Day 0 to 3	127	93	67	90	85	121	25.3	0.62	0.85	0.06
Day 3 to 52	218	270	243	158	226	194	31.0	0.08	0.03	0.95
Day 5 to 72	285b	327a	307ab	210b	347a	301ab	33.0	0.01	0.38	0.20
Day 0 to 7	198	211	186	144	200	193	26.4	0.29	0.28	0.31
Day 7 to 14	529	586	444	416	561	525	101.1	0.14	0.67	0.19
Day 0 to 14	363	398	314	280	380	358	57.0	0.12	0.49	0.17
Average daily gain, g/d
Day 0 to 3	64	5	−31	−12	−19	16	49.9	0.65	0.59	0.32
Day 3 to 52	204b	310a	396a	54b	243a	171a	58.5	<0.01	<0.01	0.15
Day 5 to 73	312	314	259	169b	380a	368ab	35.5	0.09	0.94	0.06
Day 0 to 7	172	177	155	56	179	137	39.5	0.09	0.08	0.15
Day 7 to 14	355	414	428	372	385	451	60.3	0.73	0.56	0.41
Day 0 to 14	266	307	271	244	279	296	35.5	0.41	0.73	0.58
Average daily zinc intake, mg/d
Day 0 to 3	20b	127a,b	169ax	14b	117b	310a,y	43.0	<0.01	0.20	0.07
Day 3 to 5	33c	377b	625a	21c	315b	497a	55.9	<0.01	0.11	0.49
Day 5 to 7	43c	459b	795a	28c	488b	779a	58.9	<0.01	0.98	0.88
Day 0 to 7	30c	294b	479a	20c	280b	498a	47.0	<0.01	0.96	0.90
Day 7 to 14	75c	831b	1,152a	59c	790b	1,187a	98.6	<0.01	0.86	0.87
Day 0 to 14	54c	565b	816a	41c	536b	818a	59.4	<0.01	0.70	0.94

¹Reported estimate derived from the interaction model Zn × Challenge and the covariate of BW on d 0 and sex.

²Comparisons derived from the additive model.

³Comparisons derived from the interaction model.

^a,b,cIndicate difference (P ≤ 0.05) between dietary Zn concentrations within challenge group.Indicate difference (P ≤ 0.05) between dietary Zn concentrations within challenge group.

Values are least-square means with the standard error of mean (SEM).

The ADG during d 0 to 3 was similar for the six groups (Table 1). The challenge reduced (P < 0.01) the ADG during d 3 to 5. Moreover, the ADG during d 3 to 5 was greater (P < 0.01) with a dietary Zn content of 1,400 or 2,500 than with 150 ppm, but there was no interaction between challenge and dietary Zn content (Table 1). The ADG during d 5 to 7 was influenced by an interaction (P = 0.03) between dietary Zn content and challenge. The unchallenged control pigs had similar ADG, but challenged pigs fed 150 ppm dietary Zn had a lower (P ≤ 0.05) ADG than those fed 1,400 ppm or 2,500 ppm. Moreover, pigs fed 150 ppm dietary Zn tended (P = 0.07) to have a lower ADG during d 5 to 7 when challenged with ETEC. The challenge tended (P = 0.08) to reduce the ADG during d 0 to 7, and there was also a tendency (P = 0.09) for effect of dietary Zn content. The ADG during d 7 to 14 and 0 to 14 were not affected by dietary Zn content or challenge.

Diarrhea

A three-way interaction between dietary Zn content, challenge, and day affected (P = 0.05) the feces DM percentage (Table 2). The feces DM percentage was lower (P ≤ 0.09) on d 3, 5, 7, and 14 compared to d 0 for all pigs except challenged pigs fed 1,400 ppm dietary Zn, where it was similar throughout the study. The fecal DM on d 5 was reduced (P < 0.01) from 20.9% to 13.2% for the challenge for pigs fed 150 ppm Zn. Moreover, the fecal DM on d 5 was lower (P < 0.01) for challenged pigs fed 150 ppm Zn compared to challenged pigs fed 1,400 or 2,500 ppm Zn (13.2%, 24.2%, and 23.9%, respectively).

Table 2.

Effect of dietary zinc (Zn) concentration (ppm) and challenge on fecal dry matter (%) on different days (n = 12)^1,2

Zn, ppm	Control			Challenge
	1503	1,4003	2,5003	1504	1,400	2,5005	SEM
Day 0	35.6	32.2x	31.5	33.2	26.3y	31.2	2.12
Day 3	22.7	23.7	24.5	25.7	26.0	23.2	2.38
Day 5	20.9x	23.8	20.9	13.2b,y	24.2a	23.9a	2.11
Day 7	23.2	24.3	21.6	20.4	23.7	25.9	2.02
Day 14	23.7	23.8	22.7	25.0	21.5	24.2	1.93

¹Reported estimate derived from the interaction model with Zn × challenge × day and the covariate of BW on d 0 and sex.

² P-values: Zn = 0.88, challenge = 0.52, day < 0.01, challenge × day = 0.29, Zn × challenge = 0.33, Zn × day < 0.01, Zn × challenge × day = 0.05.

³Lower (≤0.04) on d 3, 5, 7, and 14 compared to d 0, with no difference between d 3, 5, 7, and 14.

⁴Lower (≤0.04) on d 3, 5, 7, and 14 compared to d 0, lower (< 0.01) on d 5 than on d 3, greater (<0.01) on d 14 than on d 5, no difference between d 3, 7, and 14.

⁵Lower (≤0.04) on d 3, 5, and 14 compared to d 0, no difference between d 3, 5, 7, and 14, and no difference between d 0 and 7.

^abIndicate difference (P ≤ 0.05) between dietary Zn concentrations within challenge group and day.

^xyIndicate difference (P ≤ 0.05) between challenge groups within dietary Zn concentration and day.

Values are least-square means with the standard error of mean (SEM)

The diarrhea probability during wk 1, 2, and 1 to 2 was influenced by an interaction (P < 0.01) between dietary Zn content and challenge (Figure 1). The challenge increased (P < 0.01) the diarrhea risk during wk 1 and 1 to 2 for pigs fed 150 ppm, whereas challenge reduced (P < 0.03) the diarrhea risk during wk 2 and wk 1 to 2 for pigs fed 2,500 ppm dietary Zn. The diarrhea risk during the wk 1, 2, and wk 1 to 2 was found to be higher (P < 0.01) for challenged pigs fed 150 compared to those fed 1,400 or 2,500 ppm dietary Zn.

Figure 1.

Effect of dietary zinc (Zn) and challenge (C) on diarrhea probability during the first and second week, and in weeks 1 to 2 (n = 12). The obtained values are back-transformed estimated marginal means with 95% confidence intervals. ^abIndicate difference (P ≤ 0.05) between Zn concentrations within challenge group. ^xyIndicate difference (P ≤ 0.05) between challenge groups within Zn concentration.

Shedding of ETEC F18 and toxins

The fecal shedding of ETEC F18 was affected by interactions between dietary Zn content interacted with challenge (P = 0.02), and challenge interacted with day (P < 0.01; Figure 2A). The shedding of LT was influenced by a three-way interaction between dietary Zn content, challenge, and day (P < 0.01; Figure 2B). Lastly, two-way interactions affected the shedding of STb; challenge interacted with day (P < 0.01), and dietary Zn interacted with day (P < 0.01; Figure 2C). The ETEC challenge increased (P < 0.01) the shedding of ETEC F18, STb, and LT on d 3, 5, and 7 for all dietary Zn concentrations (Figure 2); and the F18 shedding on d 14 for pigs fed 150 or 1,400 ppm dietary Zn (P < 0.03). Challenged pigs fed 150 compared to 1,400 and 2,500 ppm dietary Zn had a greater (P ≤ 0.04) F18 and LT shedding on d 3, 5, and 7, whereas no effect of dietary Zn content was observed with the unchallenged control pigs. On d 14, the F18 shedding remained elevated (P ≤ 0.04) for challenged pigs fed 150 compared to 1,400 or 2,500 ppm dietary Zn. The STb shedding was also greater (P ≤ 0.05) for pigs fed 150 ppm dietary Zn compared to 1,400 or 2,500 ppm dietary Zn for both control and challenged pigs throughout the study (Figure 2).

Figure 2.

Effect of dietary zinc (Zn) and challenge (C) on fecal shedding of (A) ETEC F18, (B) LT toxin, and C) STb toxin on days (D) 0, 3, 5, 7, and 14 (n = 12). Estimated marginal means with 95% confidence intervals are shown. ^abIndicate difference (P ≤ 0.05) between Zn concentrations within challenge group and day. ^xyIndicate difference (P ≤ 0.05) between challenge groups within Zn concentration and day. The comparison between days within the Zn concentration and challenge group is deliberately not shown to increase the readability of the figure.

The comparisons between days within the dietary Zn concentration and challenge group are deliberately not shown in Figure 2 to improve the readability. Compared to d 0, the fecal ETEC F18 shedding in challenged pigs was higher (P < 0.05) on d 3, 5, and 7. Whereas the shedding in control pigs was lower (P < 0.01) on d 14. Similarly, LT shedding on d 3, 5, and 7 was greater (P < 0.01) compared to d 0 for challenged pigs. In addition, the LT shedding on d 5 was greater (P < 0.01) for challenged pigs fed 150 ppm dietary Zn compared to the shedding level on d 3. The LT shedding was increased (P < 0.01) on d 14 compared to the initial level on d 0 for control pigs fed 1,400 ppm dietary Zn. The shedding of STb toxin on d 3, 5, and 7 was greater (P < 0.01) compared to d 0 for challenged pigs fed 150 ppm dietary Zn. In contrast, STb shedding in control pigs fed 2,500 ppm dietary Zn was lower (P < 0.01) on d 3, 5, 7, and 14 compared to d 0, and the shedding was also reduced (P < 0.01) on d 14 compared to d 0 in control pigs fed 1,400 and challenged pigs fed 1,400 or 2,500 ppm dietary Zn.

The ADG during d 3 to 5 was negatively correlated (P < 0.03) with F18, STb, and LT fecal shedding on d 3 (R² = 0.19, 0.15, and 0.22, respectively). On d 5, only STb shedding was negatively (P = 0.05) correlated with ADG during d 5 to 7 (R² = 0.06).

Moreover, fecal DM percentage on d 3 was positively correlated (P = 0.03, R² = 0.12) with F18 shedding on d 3. On d 5 and 7, the fecal DM percentage was negatively correlated (P ≤ 0.02) with F18, STb, and LT shedding (R² = 0.09 to 0.11, R² = 0.06 to 0.12, and R² = 0.08 to 0.18, respectively). No correlations between fecal DM percentage and shedding (F18, LT, and STb) were observed on d 14.

Acute phase proteins in plasma

The CRP concentration tended (P = 0.08) to be influenced by a two-way interaction between challenge and day, haptoglobin tended (P = 0.08) to be affected by a three-way interaction between dietary Zn content, challenge, and day, while no interactions were observed for the pig-MAP concentration (Table 3). The ETEC challenge tended (P = 0.07) to increase the CRP concentration, while no effect of challenge was observed on the concentrations of haptoglobin or pig-MAP (Table 3).

Table 3.

Effect of dietary zinc (Zn) concentration (ppm) and challenge (C) on plasma concentration of acute phase proteins on different days (D; n = 12)¹.

	Control			Challenge			P-values
Zn, ppm	150	1,400	2,500	150	1,400	2,500	Zn	C	D	Zn × C	Zn × D	C × D	Zn × C × D
CRP, mg/L2							0.03	0.07	<0.01	0.87	0.16	0.08	0.78
Day 0	5.3p	4.7	4.2op	6.0p	8.4	5.8op
Day 3	16.0o,a	4.4b	2.4pq,b	14.5o,a	5.7b	7.0pq,b
Day 5	17.8o	7.5	7.8 op	16.8o	7.0	9.8op
Day 7	17.1op	7.9	12.5o	13.3op	13.7	11.2o
Day 14	4.8op,a	1.9ab	1.5q,b	18.1op,a	8.2ab	3.7q,b
Haptoglobin, mg/mL3							0.40	0.57	<0.01	0.25	0.36	0.99	0.08
Day 0	1.68p	0.93p	1.41p	0.97p	1.42p	1.66p
Day 3	2.89op	2.01o	2.55o	2.07o	2.57o	2.85o
Day 5	3.08o	2.38o	2.31o	2.53o	2.44o	3.13o
Day 7	2.67pq	2.39o	2.39o	2.53o	2.10op	2.78o
Day 14	1.80pq	1.74op	1.72op	2.39o	1.90op	2.47p
Pig-MAP, g/L4							0.32	0.27	<0.01	0.93	0.98	0.18	0.17
Day 0	0.94p	0.73p	0.76p	0.83p	0.79p	0.80p
Day 5	1.04o	0.97o	0.84o	1.29o	0.91o	0.16o
Day 7	0.97o	0.92o	0.90o	1.35o	1.05o	1.12o

¹Reported estimate derived from the interaction models with Zn × C × D and the covariates of BW on d 0 and sex.

²Comparisons derived from the model with C × D, D, C, and Zn as fixed effects.

³Comparisons derived from the model with Zn × C × D as fixed effects.

⁴Comparisons derived from the model with D as fixed effect.

^opqIndicate difference (P ≤ 0.05) within the column (comparison between the days within group).

^abIndicate difference (P ≤ 0.05) between Zn concentrations (150, 1,400, and 2,500 ppm) within challenge group.

Data are represented as back-transformed means. To increase the readability of this table the 95% confidence intervals can be seen in Supplementary Table S1.

On d 3, 150 ppm dietary Zn led to a greater (P = 0.01) CRP concentration compared to 1,400 or 2,500 ppm dietary Zn. On d 14, 150 ppm dietary Zn led to a greater (P = 0.03) CRP concentration compared to 2,500 ppm dietary Zn. Pigs fed 1,400 ppm dietary Zn had similar plasma CRP concentrations throughout the study, while pigs fed 150 ppm dietary Zn had elevated CRP concentration on d 3 and 5 compared to d 0. Additionally, pigs fed 2,500 ppm dietary Zn had a lower (P ≤ 0.03) CRP concentration on d 14 compared to d 0, 5, and 7.

No effect of dietary Zn on the concentration of haptoglobin or pig-MAP was observed (Table 3). The haptoglobin concentration was greater (P ≤ 0.02) on d 3 and 5 compared to d 0 in all pigs. Furthermore, the haptoglobin concentration on d 7 was greater (P ≤ 0.04) compared to d 0 in control pigs fed 1,400 or 2,500 ppm dietary Zn and challenged pigs fed 150 or 2,500 ppm Zn. On d 14, only challenged pigs fed 150 ppm Zn showed an elevated (P < 0.01) haptoglobin concentration compared to d 0. The concentration of pig-MAP was greater (P ≤ 0.06) on d 5 and 7 compared with d 0.

Serum zinc concentration

The average serum Zn concentration on d 0 was 586 ± 36.6 µg/L in all pigs (Figure 3). The serum Zn concentration was influenced by interactions between dietary Zn content and challenge (P < 0.01), and dietary Zn content and day (P < 0.01). Pigs fed 2,500 ppm Zn showed lower (P ≤ 0.03) serum Zn concentration on d 5, 7, and 14 when exposed to the ETEC challenge, while pigs fed 1,400 ppm Zn tended to have a greater (P = 0.08) serum Zn concentration on d 7 when exposed to ETEC. No effect of the ETEC challenge was observed for pigs fed 150 ppm Zn.

Figure 3.

Effect of dietary zinc (Zn) and challenge (C) on serum zinc concentration on the day (D) 0, 5, 7, and 14 (n = 12). Estimated marginal means with 95% confidence intervals are shown. ^abcIndicate differences (P ≤ 0.05) between Zn concentrations within challenge group and day. ^xyIndicate differences (P ≤ 0.05) between the challenge groups within Zn concentration and day.

The serum Zn concentrations on d 5, 7, and 14 were the lowest (P < 0.01) for pigs fed 150 ppm Zn. On d 5 and 7, challenged pigs fed 1,400 and 2,500 ppm Zn had similar serum Zn concentrations. Unchallenged control pigs fed 2,500 ppm Zn showed higher (P < 0.01) serum Zn concentrations than control pigs fed 1,400 ppm Zn. On d 14, the highest (P < 0.01) serum Zn concentration was observed with 2,500 ppm dietary Zn, regardless of challenge.

Pigs fed 1,400 or 2,500 ppm Zn had a higher (P ≤ 0.07) serum Zn concentration on d 5, 7, and 14 compared to d 0, while no changes were detected when feeding 150 ppm Zn.

The serum Zn concentration was positively correlated (P < 0.01, R = 0.84) with ADZnI. Challenge had a negative effect (P = 0.04) on the slope between ADZnI and serum Zn concentration since the slope for challenged pigs was 1.36 compared with 1.52 for unchallenged control pigs.

Pigs with diarrhea (fecal DM ≤ 18%) tended to have a lower (P = 0.08) serum Zn concentration on d 7 than non-diarrheic pigs (739 vs. 1153 µg/L). However, no correlation between serum Zn concentration and diarrhea was observed on d 5 and 14.

Discussion

In the present study, the ETEC challenge was intended to induce diarrhea and impair growth without increasing mortality. The pigs were inoculated twice rather than three times, as it is our experience that the third inoculation increases the mortality because of dehydration or a BW loss of more than 15% of the initial BW. The ETEC challenge reduced the ADG during d 3 to 5, and there was a tendency of a reduced ADG during d 0 to 7 and d 5 to 7 for pigs fed 150 ppm Zn. It was expected that the challenge would only reduce ADG temporarily based on other studies with ETEC F4 and F18 inoculation under controlled experimental settings (Madec et al., 2000; Hansen et al., 2022a). The fact that the challenge only resulted in a temporal reduced growth coincides with challenge not inducing a response in acute phase proteins in the present study, indicating the pigs rapidly recover from the challenge. Other studies have found the growth to be impaired for a longer period due to higher challenge doses or a higher challenge status in commercial farms (Madec et al., 2000; Cornelison et al., 2018). However, as reported by Madec et al. (2000), higher inoculation doses can lead to a more pronounced growth check and increased mortality.

Performance

It is well-known that dietary Zn content impacts the growth of newly weaned pigs (Poulsen, 1995; Schell and Kornegay, 1996; Hansen et al., 2022b). The present results for unchallenged control pigs showed similar patterns for the effect of dietary Zn content on ADFI during d 5 to 7 and ADG during d 3 to 5 as found by Hansen et al. (2022b). ADFI (d 5 to 7) and ADG (d 3 to 5) were greater with 1,400 ppm dietary Zn than 150 ppm dietary, and 2,500 ppm dietary Zn led to either similar or greater ADFI and ADG than 150 ppm dietary Zn. A similar pattern in the effect of dietary Zn concentration on ADG during d 5 to 7 was observed in challenged pigs, thus, indicating that challenged pigs respond more sensitively in ADG to dietary Zn content than unchallenged controls. Overall, there was no beneficial effect on performance of feeding newly weaned pigs 2,500 ppm Zn compared to 1,400 ppm dietary Zn for either control or challenged pigs.

Diarrhea

The challenge resulted in an increased risk of diarrhea and feeding pigs 150 ppm Zn aggravated the diarrhea caused by the ETEC challenge. This corresponds to the findings of others (Madec et al., 2000; Lei and Kim, 2020). Our results also correspond to Wang et al. (2022), who found that diarrhea induced by ETEC F4 was aggravated by feeding mice a marginal Zn-deficient diet. The underlying mechanism behind this could perhaps be, that Zn deficiency combined with the ETEC F4 challenge resulted in a reduction in the expression of the tight junction proteins, which increases the intestinal permeability leading to diarrhea (Wang et al., 2022). However, there was no difference in fecal DM on d 5 or diarrhea probability between challenged pigs fed 1,400 or 2,500 ppm dietary Zn, indicating no beneficial effect of increasing the dietary Zn from 1,400 to 2,500 ppm dietary Zn.

ETEC shedding

In the present study, the ETEC challenge increased the shedding of ETEC F18 and toxins in feces during the first week, which is similar to results from other ETEC F18 challenge studies (Kwon et al., 2014; Hansen et al., 2022a). Boeckman et al. (2022) observed a greater ETEC 62-57nal number on the mucosal surface in the duodenum and in the intestinal contents of the cecum and colon in pigs challenged with ETEC 62-57nal, which indicates a correlation between the presence of ETEC in feces and colonization in the small intestine. The fecal shedding of ETEC F18 and toxins were reduced by 1,400 compared to 150 ppm dietary Zn. This could perhaps be due to the inhibitory effect of Zn on bacterial adhesion and biofilm formation (Roselli et al., 2003; Wu et al., 2013) and/or the positive impact of Zn on the integrity of the intestinal barrier function, including the production of mucin (Maares et al., 2020), which may inhibit ETEC colonization (Dean-Nystrom and Samuel, 1994). Increasing the dietary Zn concentration from 1,400 to 2,500 ppm in the ETEC-challenged pigs had no additional inhibitory effect on ETEC and toxin shedding, therefore, it does not seem necessary to provide newly weaned pigs more than 1,400 ppm dietary Zn. Together, the results from the present study indicate that diets with 150 ppm Zn as compared to 1,400 or 2,500 ppm Zn cause greater ETEC shedding and an increased risk of diarrhea.

The challenge increased the shedding of ETEC and toxins, and the ADG was negatively correlated to these during the acute phase of the challenge (d 3 to 5 and 5 to 7), indicating that greater shedding resulted in greater negative impact on ADG. The fecal DM was also correlated with ETEC shedding, but the direction of the correlation was inconsistent over time, perhaps indicating that it takes about three days from the last ETEC inoculation for the diarrhea to typically peak (Hansen et al., 2022a).

Acute phase proteins

Although ETEC challenge and dietary Zn content affected the fecal shedding of ETEC and toxins as well as the risk of diarrhea, the challenge and dietary Zn content had, in general in the present study, no to limited impact on the concentration of acute phase proteins in plasma. It was expected that challenge would increase acute phase proteins, as it has previously been shown that ETEC F18 challenge causes an increase in the concentration of e.g., CRP (Hansen et al., 2022a). Moreover, Zn has an anti-inflammatory effects (Prasad, 2008), but only CRP concentration in the present study was decreased by high dietary Zn concentrations (1,400 and 2,500 ppm) on d 3 and 14. The fact that we observed no effect of dietary Zn on haptoglobin in the present study contradicts Bergeron and Guay (2019), who showed that 2,500 ppm dietary Zn reduced the haptoglobin concentration on d 8 and 15 after weaning compared to 100 ppm dietary Zn. The lack of response in acute phase proteins to the ETEC challenge could be because the acute phase proteins response is sensitive to their pre-challenge concentration, which is influenced by age, stress, and housing conditions (Petersen et al., 2002; Pomorska-Mól et al., 2012). This study would have gained a greater understanding of the ETEC challenge impact on the inflammatory response if other inflammatory responses were measured, such as for example the concentration of pro-inflammatory cytokines in the small intestinal mucosa. The concentration of interleukin-6 and interleukin-8 in the small intestine’s mucosa could be a potentially better biomarker for inflammation in the small intestine caused by ETEC F18 (McLamb et al., 2013).

Serum zinc concentration

Hansen et al. (2022b) showed that maintaining the serum Zn concentration one week after weaning to the serum Zn concentration at the time of weaning (767 µg/L) required 1,121 ppm dietary Zn, which the present results contradict, as the weaning serum Zn concentration (586 µg/L) could be maintained with 150 ppm dietary Zn. However, the initial serum Zn concentration in the present study was lower than in Hansen et al. (2022b), which could have influenced the serum Zn response to dietary Zn content. Moreover, Hansen et al. (2022b) found that the risk of diarrhea increased up to 60% when the serum Zn concentration was below 767 µg/L. The present study showed that pigs with diarrhea (fecal DM ≤ 18%) had a lower serum Zn concentration, indicating that either diarrhea in itself might have a negative impact on the Zn status or the other way around, that low serum Zn status results in increased diarrhea. Nevertheless, feeding 150 ppm Zn to newly weaned pigs may lead to increased diarrhea, as the serum Zn concentration in the present study was below 767 µg/L with 150 ppm dietary Zn, and ETEC challenge had a greater impact on the diarrhea for pigs fed 150 ppm dietary Zn.

Earlier studies have demonstrated a decrease in serum Zn concentration in response to E. coli infection (Tufft et al., 1988; Erskine and Bartlett, 1993; Eijkelkamp et al., 2019). The challenge in the present study only decreased the serum Zn concentration in pigs fed 2,500 ppm Zn. Wang et al. (2022) observed a similar trend in mice challenged with ETEC F4 as the serum Zn concentration was numerically reduced in response to challenge for mice fed a Zn-adequate diet, while no change was observed for mice fed a Zn-deficient diet. It has been suggested that Zn deficiency may cause a redistribution in the body´s Zn pools in response to infection, because of an altered pattern in cytokine production (Beck et al., 1997; Wang et al., 2022). It is therefore possible that an ETEC challenge affects the serum Zn concentration differently depending on the Zn status (deficient vs adequate). This could explain why 150 ppm dietary Zn led to no significant reduction in the serum Zn concentration, as pigs were marginally Zn deficient throughout the study (their serum Zn concentration was below 700 µg/L; Puls, 1990; Hotz et al., 2003; Hess et al., 2007). It is uncertain why the ETEC challenge did not affect the serum Zn concentration in pigs fed 1,400 ppm Zn, as their serum Zn concentration was above 700 µg/L during the entire study period. However, the regulation of the Zn absorption mechanisms (passive and active) might be involved. The active absorption mechanism decreases in response to increasing Zn concentration in the digesta, while the passive absorption mechanism is gradient-determined (Condomina et al., 2002; Karweina et al., 2015). Dalto et al. (2023) found that dietary Zn on d 2 after weaning did not influence the expression of Zn transporter 4 (ZIP4) in the small intestine, transporting Zn from the lumen into the enterocyte. However, two weeks after weaning, the ZIP4 expression was lower with 3,000 than 1,000 ppm dietary Zn, and these dietary Zn contents resulted in lower expression than 150 ppm dietary Zn. In contrast, passive absorption increases with increasing dietary Zn content (Steel and Cousins, 1985). Generally, it is hypothesized that deficient to adequate Zn supply leads to mainly active Zn absorption, while adequate to excessive Zn supply mainly depends on passive Zn absorption (Martin et al., 2013). It is known that ETEC influences the absorption of Zn (and other nutrients) because ETEC negatively affects enterocyte tight junctions, which lead to increased permeability (Wang et al., 2022), and may reduce small intestinal expression of ZIP4 (Jang et al., 2023). However, it requires further research to determine the impact of an ETEC F18 challenge on Zn absorption in pigs fed increasing dietary Zn concentrations. It would also be interesting to look into the effects of an ETEC challenge and dietary Zn content on the concentration of cytokines and Zn in different tissues (e.g., liver, mucosa, and serum), as well as on small intestinal expression of tight junction and transport proteins.

In conclusion, the ETEC F18 challenge resulted in greater ETEC F18 shedding in feces and diarrhea but it was not severe enough to cause a response in the acute phase proteins (CRP, haptoglobin, and pig-MAP). The results of this study indicate that newly weaned pigs fed 150 ppm Zn were more susceptible to ETEC F18 colonization and its negative consequences, as they had reduced ADG during d 5 to 7, the highest fecal shedding of ETEC and toxins, and highest diarrhea risk. As there was no difference in performance, diarrhea risk, or fecal shedding of ETEC and toxins between 1,400 and 2,500 ppm dietary Zn, the results indicate that 1,400 ppm dietary fulfilled the Zn requirement for pigs challenged with ETEC F18.

Glossary

Abbreviations

ADFI

average daily feed intake

ADG

average daily gain

ADZnI

average daily zinc intake

BW

body weight

CRP

C-reactive protein

DM

dry matter

ETEC

Enterotoxigenic Escherichia coli

LT

heat-labile enterotoxin

Pig-MAP

pig major acute protein

qPCR

quantitative polymerase chain reaction

STb

heat-stable enterotoxin b

Conflict of interest statement

The authors declare no conflict of interest.