Impacts of antibiotics on weanling pigs

Trace amounts may exacerbate disease in pigs

By Kwangwook KimBesides the evolving threat of antibiotic resistance, the emergence of trace levels of antibiotics in the environment has escalated as one of the top public health challenges. It has been reported that a wide variety of trace levels of veterinary antibiotics have been detected in different on-farm environments, including surface water, soil, air and dust. Exposure to trace levels of antibiotics may not only increase the emergence of antibiotic-resistant bacteria but also cause adverse health effects on animals and humans.The potential detrimental effects caused by exposure to trace amounts of antibiotics include toxicity, mutagenicity, carcinogenicity, reproductive disorders, allergy, and the pharmacokinetics of infectious diseases in organisms. Especially, young animals that are highly susceptible to diseases are more sensitive to trace amounts of antibiotics due to their immature immune system and gut microbiota community.However, limited studies have evaluated the impacts of trace amounts of antibiotics and underlying mechanisms on young pigs, particularly under disease-challenged conditions.Therefore, the studies were conducted to investigate the impacts of trace amounts of antibiotics on health of weanling pigs experimentally infected with enterotoxigenic E. coli (ETEC) F18, which is one of the predominant strains in the United States that induces post-weaning diarrhea.Impacts of trace amounts of antibiotics on weanling pigs
Thirty-four weanling pigs were assigned randomly to three dietary treatments: 1) the complex nursery basal diet (control; CON), 2) addition of 0.5 mg/kg carbadox (trace amounts of antibiotic; TRA) to the basal diet, or 3) addition of 50 mg/kg carbadox (label-recommended dose of antibiotic; REC) to the basal diet.Spray-dried plasma, other antibiotics except carbadox, and high levels of zinc oxide exceeding recommendation and normal practice were not included in the diets.After seven day of adaptation, all pigs were orally inoculated with 3 mL of ETEC F18 (1010 colony-forming unit (CFU) per 3 mL dose in phosphate-buffered saline) for three consecutive days, which induced mild diarrhea.To assess the health status of pigs, growth performance, severity of diarrhea, and local and systemic inflammatory markers were measured. Pigs fed TRA had the lowest (P < 0.05) average daily gain and feed efficiency from day 0 to 5 post-infection (PI), near the peak infection period, compared with pigs in the other groups (Table 1).Compared with pigs in the CON and TRA groups, pigs fed REC had lower (P < 0.05) average diarrhea scores from day 0 to 5 PI and day 5 to 11 PI (recovery period) and frequency of diarrhea (Table 2). Pigs fed TRA had the greatest (P < 0.05) serum C-reactive protein on days 2 and 5 PI, and serum TNF-α on day 5 PI, compared with pigs in the CON and REC groups.Pigs in the TRA group had the highest (P < 0.05) bacterial translocation in lymph nodes on day 11 PI compared with pigs in the other groups. Pigs fed TRA upregulated (P < 0.05) mRNA expression of IL1B, IL6, and COX2 in ileal mucosa on day 11 PI compared with the REC. These results can be explained by trace amounts of antibiotics exacerbated the detrimental effects of ETEC infection on pig performance by increasing diarrhea and systemic inflammation of weanling pigs.To decipher the detrimental effects of trace amounts of antibiotics, the metabolomics and microbiome approaches were used. Based on the identified metabolites in serum and distal colon digesta, a PLS-DA score plot with 95% confidence ranges (shaded area) showed a clear separation between the TRA and REC groups near the peak infection and recovery period (Figure 1).Pathway analysis and metabolite set enrichment analysis indicated that trace amounts of antibiotics enhanced amino acid depletion and carbohydrate catabolism. Moreover, purine metabolisms were upregulated in TRA due to activated immune cells. Microbial profiles in distal colon digesta indicated that compositional differences were observed between CON vs. REC and TRA vs. REC on days 5 and 11 PI (Pairwise-Adonis, P < 0.05) (Figure 2).Within the Firmicutes phylum, pigs in the TRA group had lower (P < 0.05) relative abundance of Lactobacillaceae (8.91% vs. 21.33%) than pigs in REC on day 5 PI, whereas REC had lower (P < 0.05) relative abundance of Lactobacillaceae (5.82% vs. 23.90% or 27.69%) than pigs in the CON or TRA groups on day 11 PI. Pigs in the REC group had higher (P < 0.05) relative abundance of Clostridiaceae (17.14% vs. 1.45%) and Streptococcaceae (10.09% vs. 0.21%), but lower (P < 0.05) relative abundance of Lachnospiraceae (20.25% vs. 27.44%) in the distal colon on day 11 PI than on day 5 PI.The impacted metabolic pathways and colonic microbial shift may also be closely associated with the slow growth and delayed recovery from ETEC infection of weaned pigs supplemented with trace amounts of antibiotics.In conclusion, the deterioration of disease caused by trace amounts of antibiotics was likely due to the increased severity of local and systemic inflammation of pigs. Moreover, as a consequence of alteration of metabolic pathways and colonic microbial shift, more nutrients were used for immune responses instead of supporting the growth of pigs in trace amounts of antibiotics group.Further research will consider repeating the ETEC challenge study with a larger number of weaned pigs in order to confirm the detrimental effects of trace amounts of antibiotics in animal feed. Additionally, targeted metabolomics and metagenomics could provide more insights into the potential risk of trace amounts of antibiotics on the host response to ETEC infection.

Figure 1. Partial Least Squares Discriminant Analysis (PLS-DA) 2D score plot of the metabolites in serum (A, B) or colon digesta (C, D) showed separated clusters between the TRA and REC groups on day 5 (A, C) and 11 (B, D) post-infection. Red = CON (Control); Blue = TRA (Trace amounts of antibiotic); Green = REC (Label-recommended dose of antibiotic). Shaded areas in different colors represent in 95% confidence interval.

Figure 2. Beta diversity (Adonis analysis based on the Bray-Curtis distance) of colon digesta of ETEC F18 challenged pigs fed diets supplemented with different dose of antibiotic on day 5 and 11 post-infection. Data were analyzed by principal coordinate analysis.

Item¹	Diet CON²	TRA	REC⁴	SEM	P value
BW, kg
day -7	6.79	6.72	6.89	0.27	0.94
day 0	8.56	8.93	8.68	0.39	0.85
day 5 PI	10.54^ab	10.16^ab	11.89^a	0.41	0.08
day 11 PI	14.29^b	13.89^b	17.06^a	0.52	<0.05
ADG, g
day -7 to 0	251	314	284	31.76	0.50
day 0 to 5 PI	409^a	232^b	489^a	72.59	<0.05
day 5 to 11 PI	608	651	675	44.38	0.37
ADFI, g
day -7 to 0	425	449	349	48.20	0.29
day 0 to 5 PI	620	527	648	48.53	0.36
day 5 to 11 PI	856	833	897	44.09	0.24
G:F
day -7 to 0	0.61	0.74	0.81	0.072	0.24
day 0 to 5 PI	0.64^a	0.37^b	0.73^a	0.099	<0.01
day 5 to 11 PI	0.70	0.79	0.69	0.050	0.42
Table 1. Growth performance of ETEC F18 challenged pigs fed diets supplemented with antibiotics.
^a,bWithin a row, means without a common superscript differ (P<0.05). ¹BW = body weight, ADG = average daily feed intake, ADFI = average daily feed intake, G:F = gain:feed, and PI = post-infection. Each least square means represents 9 to 13 observations, except the day 5 to 11 that has 5 to 7 observations. ²CON = Control. ³TRA = Trace amounts of antibiotic. ⁴REC = Label-recommended dose of antibiotic.

	Diet CON¹	TRA²	REC³	SEM	P-value
Diarrhea score
day 0-5⁴	2.87^a	2.62^a	1.94^b	0.26	<0.01
day 5-11⁵	2.59^a	2.74^a	1.48^b	0.34	<0.05
Pen days	120	84	105
Frequency of diarrhea⁶	29.17^a	26.19^a	7.62^b	-	<0.05
Table 2. Periodical diarrhea score and frequency of diarrhea of ETEC F18 challenged pigs fed diets supplemented with antibiotics.
^a,bWithin a row, means without a common superscript differ (P<0.05). ¹CON = Control. ²TRA = Trace amounts of antibiotic. ³REC= Label-recommended dose of antibiotic. ⁴Each least squares mean represents 9 to 13 observations. ⁵Each least squares mean represents 5 to 7 observations. ⁶Frequency = Number of pen days with fecal score ≥ 4.

Kim is a postdoctoral scholar in the Department of Animal Science at the University of California, Davis.