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. 2023 Apr 14;102(7):102722. doi: 10.1016/j.psj.2023.102722

Supplementation of exogenous bile acids improve antitrichomonal activity and enhance intestinal health in pigeon (Columba livia)

Hui Ma *,1, Shixiong Bian *,1, Pengmin Han †, Yunlei Li *, Aixin Ni *, Ran Zhang *, Pingzhuang Ge *, Yuanmei Wang *, Jinmeng Zhao *, Yunhe Zong *, Jingwei Yuan *, Yanyan Sun *, Jilan Chen *,2
PMCID: PMC10196791  PMID: 37167885

Abstract

The study investigated the effects of supplementation of bile acids in drinking water on antitrichomonal activity, growth performance, immunity and microbial composition of pigeon. A total of 180 pairs of White King parent pigeons were randomly assigned to 5 treatments of 6 replications with 6 pairs of parent pigeons and 12 squabs in each replicate. The control (CON) group drank water without any additions. The metronidazole (MTZ) group drank water with 500 μg/mL metronidazole for 7 d and without any additions in other days. The else groups drank water with 500, 750, and 1,250 μg/mL bile acid (BAL, BAM, BAH) for 28 d. The results showed that Trichomonas gallinae (T. gallinae) in MTZ, BAL, BAM, and BAH groups were lower than that in CON group at 14, 21, and 28 d of parent pigeons (P < 0.05) and at 21 and 28 d of squabs (P < 0.05). Albumin and alanine transaminase in CON group were higher than those in MTZ, BAL, and BAH groups (P < 0.05). The levels of soluble CD8 were higher in MTZ and BAH groups compared with CON group (P < 0.05). The lesions in oral mucosa, thymus, liver, and spleen tissues of CON group could be observed. Abundance-based coverage estimator (ACE) index in BAH group was higher than that in CON and MTZ groups. Simpson index in CON and BAH groups was higher than MTZ group (P < 0.05). Lactobacillus was the highest colonized colonic bacteria in genera that were 77.21, 91.20, and 73.19% in CON, MTZ, and BAH, respectively. In conclusion, drinking water supplemented with 500, 750, and 1,250 μg/mL bile acid could inhibit growth of T. gallinae in both parent pigeons and squabs. Squabs infected with T. gallinae in control group had higher mortality rate and more serious tissue lesions. Squabs in bile acids treated group had more sCD8 in serum and abundant intestinal morphology. Bile acids could be an efficient drinking supplements to inhibit T. gallinae and improve pigeon adaptive immunity and intestinal health.

Key words: bile acid, Trichomonas gallinae, antitrichomonal activity, intestinal health, pigeon

INTRODUCTION

Trichomonas gallinae (T. gallinae) is a flagellated protozoan parasite responsible for trichomoniasis. The parasite infects the anterior digestive and occasionally respiratory tracts of a large variety of birds (Rouffaer et al., 2014; Tabari et al., 2017). The disease is commonly seen as a caseous lesion within the anterior region of the digestive tract. The lesions vary from mild to severe clinical infections, and the severe fatal inflammation can kill the birds by obstructing the esophageal lumen (Gerhold et al., 2008; Stockdale et al., 2015). Trichomoniasis is a worldwide epidemic parasitic disease and considered to be a major contribution of wide ranging mortality in pigeon, especially the young birds (Lawson et al., 2011; Seddiek et al., 2014). Captive pigeons are more susceptible than the wild birds. Mckeon reported the prevalence of T. gallinae was 49% in captive pigeons and 46% in wild birds in Australia (Mckeon et al., 1997). The differences in prevalence of T. gallinae were more remarkable as 35% in wild pigeons and 68% in captive birds in Saudi Arabia (Borji et al., 2011).

Metronidazole, most commonly used for curing trichomoniasis, is effective against wide range of protozoal and anaerobic bacterial pathogens (Aydın et al., 2000; Seddiek et al., 2014; Tabari et al., 2017). Metronidazole is well absorbed and distributed in tissues, including the central nervous system, placenta, and bone (Steinman et al., 2000; Świtała et al., 2016), and the side effects of metronidazole intakes are serious, as transient neutropenia, nausea, cancer, and peripheral neuropathy (Kurohara et al., 1991; Sobel et al., 1999). Thus, looking for nonchemical and natural equivalents is essential to treat T. gallinae infections. The plant extracts and traditional medicines have been used in trichomoniasis treatment and prevention, but there are also some defects as ineffective and high cost (Woronuk et al., 2011; Malekifard et al., 2021).

Bile acids are a peculiar family of amphipathic molecules derived from cholesterol through a series of enzymatic reactions (Hofmann and Hagey, 2008). Bile acids are generated in the liver, transported through the biliary system, and stored in the gall bladder (Gándola et al., 2020). Bile acids, secreted into the intestine with nutrients flow when taking food, participate in fat solubilization and absorption in the gut (Fiorucci et al., 2018). Most of the bile acids are reabsorbed in the gut and recycled back to the liver via the portal vein by enterohepatic circulation (Fiorucci et al., 2018). However, many other functions of bile acids have been described recently. All bile acids are signaling molecules by activating a family of evolutionary conserved receptors in nuclear (farnesoid X receptor, FXR) and cell surface (G protein coupled bile acid receptor, GPBAR1) collectively known as bile acids activated receptors (BARs) (Chiang, 2009; Copple and Li, 2016). Consequently, bile acids participate in different biological processes, including lipid metabolism, glucose homeostasis, energy expenditure, antiviral immunity, and immune cell function (Šarenac and Mikov, 2018; Hu et al., 2019). Bile acids have been reported to regulate various disease-related events as inflammasome activation, phagocytosis of hepatic macrophages, and viral hepatitis by binding to the Takeda G Protein Coupled Receptor-5 (TGR5) (Gadaleta et al., 2015; Perino and Schoonjans, 2015; Guo et al., 2016). Hu et al. reported that when virus infected mice, the accumulated intracellular bile acids could activate the TGR5-β-arrestin-SRC axis, improve synthesis of antiviral signaling components and initiate innate antiviral immune response (Hu et al., 2019). Bile acids and their derivatives also possess the therapeutic effects of human type of immunodeficiency virus 1 (HIV1) (Šarenac and Mikov, 2018). But there are still no reports of the therapeutic effects of bile acids in parasite.

In the current study, we investigated the antitrichomonal effect of bile acids in vivo by adding different concentration of bile acids into the drinking water comparing with the effect of metronidazole as a standard antitrichomonal drug. The effect of added bile acids on biochemical index, growth performance, gut microbiota, and other indicators of the squabs was evaluated. These findings showed new activities of bile acids in regulating disease resistance and opened therapeutic strategies for treating trichomoniasis in pigeons.

MATERIALS AND METHODS

Ethics Statement

The experimental protocol used in the present study was approved by the Animal Care and Use Committee of the Institute of Animal Sciences of Chinese Academy of Agricultural Sciences (IAS2021-97). All procedures were conducted in accordance with the institutional animal ethics guidelines set by the Ministry of Agriculture and Rural Affairs of the People's Republic of China.

Experimental Birds, Diet and Management

A total of 180 pairs of White King parent pigeons were randomly assigned to 5 treatments with 6 replications, each replicate including 6 pairs of parent pigeons and 12 squabs. All birds had free access to feed and water during the experiment. The ingredients and nutrient levels of basal diets were formulated to meet the nutrient requirements of parent pigeons (Table 1). The 5 drinking treatments of parent pigeons were as follows: 1) control (CON), 2) parent pigeons drank water containing 0.05% metronidazole (500 μg/mL, MTZ), 3) parent pigeons drank water containing 0.05% bile acid (500 μg/mL, BAL), 4) parent pigeons drank water containing 0.075% bile acid (750 μg/mL, BAM), and 5) parent pigeons drank water containing 0.125% bile acid (1,250 μg/mL, BAH). Metronidazole (purity >98%) used was obtained from Beijing Solabo Technology Co. Ltd. (Beijing, China). Bile acid (purity >20%) used was obtained from Shandong Longchang Animal Health Co. Ltd. (Shandong, China). The experiment lasted for 28 d. MTZ group drank water with metronidazole supplement for the period d 1 to d 7, while during d 7 to d 28 they drank water without addition. BAM, BAL, and BAH groups drank water with bile acid supplement during the whole period d 1 to d 28.

Table 1.

Composition and nutrient levels of the basal diet of parent pigeons.

Item Content (%)
Ingredients
 Corn 38.30
 Pea 34.75
 Wheat 5.70
 Soybean meal 14.35
 Soybean oil 2.18
 Sorghum 1.05
 CaHPO4 0.67
 Stone powder 2.00
 Premix1 1.00
 Total 100.00
Nutrient levels2
 Metabolizable energy (MJ/kg) 12.13
 Crude protein 17.43
 Calcium 0.46
 Available phosphorus 0.30
 Lysine 1.09
 Methionine 0.23
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1

Premix supplied per kilogram of diet: vitamin A, 4,100 IU; vitamin D3, 1,700 IU; vitamin E, 25 IU; vitamin K, 1 mg; niacin, 15 mg; folic acid, 0.55 mg; pantothenic acid, 7.5 mg; cuprum, 10 mg; iron, 35 mg; manganese, 56 mg; zinc, 40 mg; iodine, 0.35 mg; selenium, 0.20 mg.

2

The nutrient level of the diet was calculated.

T. Gallinae Test

Throat swab specimens were used to test the numbers of T. gallinae in oral cavity of parent pigeons and squabs. After scraping in throat, the swab specimens were put into tubes with 1 mL of normal saline. The tubes were centrifuged at 3,000 × g for 5 min. Supernatant was removed and sediment was resuspended in 100 μL normal saline. Hemocytometer was used to count the numbers of T. gallinae.

Performance Parameters

Body weights of squabs at 1, 3, 7, 14, 21, and 28 d were recorded to calculate the average daily gain (ADG). Mortalities and health status were observed and recorded daily during the experiment.

Sample Collection

At 28 d, 6 squabs of each treatment were randomly selected. Blood from the wing vein was collected in sterile vacuum blood collection tubes and kept in ice. Serum samples were obtained after centrifugation (3,000 × g for 15 min at 4°C) and stored at −80°C until analysis. The selected birds were sacrificed by jugular exsanguination. The carcass weight (without neck and feet), breast muscle, liver, heart, stomach, thymus, bursa of Fabricius, spleen, and abdominal fat were weighed after flushing with saline. The organ index of liver, heart, muscularis stomach, gland stomach, thymus, spleen, and bursa of Fabricius was calculated using the formula: organ index = organ weight (g)/body weight (g) × 100. Left breast meat was separated to measure the meat quality. The middle of mucosa of oral cavity, thymus, liver, spleen, and duodenum (1 cm) was collected, rinsed, and fixed in 4% paraformaldehyde solution for histological examination. The contents of the duodenum were collected aseptically, snap frozen, and stored at −80°C for 16S rRNA sequencing.

Serum Sample Analysis

The level of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), albumin (ALB), alanine transaminase (ALT) and aspartate transaminase (AST) in the serum was determined using commercial assay kits (Weifang Zecen Biotechnology Co. Ltd., Weifang, China). The level of immunoglobulin A (IgA), immunoglobulin G (IgG), soluble CD4 (sCD4), and soluble CD8 (sCD8) was measured with ELISA kits (Beijing Laibotairui Technology Co. Ltd., Beijing, China). All steps were carried out according to the manufacturer's instructions.

Meat Quality Analysis

The pH of the left breast meat was measured by a calibrated glass-electrode pH meter at 1 h and 24 h after slaughter (pH spear test 30, Thermo Electron Corporation, Waltham, MA). The breast meat lightness (L*), redness (a*), and yellowness (b*) values were determined (Minolta CM-700d Chromameter; Konica Minolta Sensing Inc., Osaka, Japan). Drip loss was measured according to the plastic bag method described by Honikel (1998).

Histomorphology and Analysis

Tissues fixed in paraformaldehyde solution were dealt with paraffin embedding techniques, sliced into transects (5 mm), and stained with hematoxylin and eosin to observe their histomorphology. For the duodenum tissue, the villus height (VH, from the tip of the villus to the crypt opening) and crypt depth (CD, from the base of the crypt to the crypt opening) of at least 10 well-oriented villi were measured and the ratio of VH/CD was calculated. All histomorphometry data acquisition were performed using Olympus microscope (Olympus, Tokyo, Japan) and analyzed by the ImagePro Plus 6.0 software.

Colon Microbiota Populations

Colon microbiota populations of CON, MTZ, and BAH groups were observed. Microbial DNA was extracted from colon contents using the TGuide S96 Magnetic Soil/Stool DNA Kit (Tiangen Biotechnology Co., Ltd., Beijing, China). Multiplexed 16S rRNA libraries were constructed by Illumina novaseq 6000 (Illumina, Santiago, CA) using the V3 to V4 region of 16S rRNA for target amplification (338F: 5′- ACTCCTACGGGAGGCAGCA-3′ and 806R: 5′- GGACTACHVGGGTWTCTAAT-3′). The obtained sequences were processed for alignment and cluster into operational taxonomic units (OTUs) with a 97% similarity using USEARCH (v10.0) in QIIME2 software using the SILVA database. The alpha and beta diversity were calculated by QIIME2 and R software.

Statistical Analysis

Data were expressed as means ± deviation (SD). Comparison among groups was assessed by 1-way analysis of variance (ANOVA) and Dunn's multiple comparisons using SAS statistical software (version 8.1, SAS Institute, Cary, NC). Death rates of squabs were analyzed by chi-square text with SAS. Statistical significance was set at P < 0.05.

RESULTS

Numbers of T. Gallinae in Oral Cavity

The antitrichomonal activity of bile acids was evaluated by detecting the number of T. gallinae in oral cavity. The results showed that bile acids had an efficient antitrichomonal action against trichomoniasis as metronidazole. The initial numbers of T. gallinae had no significant differences among the 5 groups (Table 2). After different treatment for 7 d, T. gallinae of parent pigeons in MTZ and BAH groups decreased to 0, which was significantly lower than that of parent pigeons in CON and BAL groups (P < 0.05). While at 14 d and 28 d after treatment, T. gallinae of parent pigeons in MTZ, BAL, BAM, and BAH groups was all significantly lower (P < 0.05) than CON group (Table 2). For squabs, the number of T. gallinae at 28 d was 5 times more than the number at 7 d for CON group (Table 3). For MTZ, BAL, BAM, and BAH groups, numbers of T. gallinae at 21 d and 28 d were all significantly lower than those of CON group (Table 3).

Table 2.

The number of T. gallinae of parent pigeons (×104/mL).

Groups1 0 d 7 d 14 d 21 d 28 d
CON 6.71 ± 3.50 4.14 ± 2.49a 3.26 ± 2.78a 3.48 ± 2.53a 6.22 ± 5.26a
MTZ 7.19 ± 2.86 0.00 ± 0.00b 0.00 ± 0.00b 0.08 ± 0.37b 0.94 ± 1.69b
BAL 6.17 ± 5.92 1.83 ± 2.47c 0.29 ± 0.96b 0.00 ± 0.00b 0.00 ± 0.00b
BAM 7.86 ± 4.27 0.19 ± 0.58b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b
BAH 7.11 ± 2.75 0.00 ± 0.00b 0.00 ± 0.00b 0.27 ± 1.46b 0.20 ± 0.81b
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a-c

Means within a column with different superscripts indicated significant difference (P < 0.05).

1

CON = control; MTZ = 0.05% metronidazole; BAL = 0.05% bile acid; BAM = 0.075% bile acid; BAH = 0.125% bile acid.

Table 3.

The number of T. gallinae of squabs (×104/mL).

Groups1 7 d 14 d 21d 28 d
CON 2.34 ± 3.60a 7.81 ± 9.29a 9.17 ± 10.42a 14.91 ± 17.03a
MTZ 0.00 ± 0.00b 0.00 ± 0.00c 0.00 ± 0.00b 1.82 ± 2.37b
BAL 0.00 ± 0.00b 0.00 ± 0.00c 0.00 ± 0.00b 2.98 ± 5.76b
BAM 0.80 ± 1.62c 2.65 ± 5.97b 0.00 ± 0.00b 0.00 ± 0.00b
BAH 0.00 ± 0.00b 0.00 ± 0.00c 0.00 ± 0.00b 0.00 ± 0.00b
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a–c

Means within a column with different superscripts indicated significant difference (P < 0.05).

1

CON = control; MTZ = 0.05% metronidazole; BAL = 0.05% bile acid; BAM = 0.075% bile acid; BAH = 0.125% bile acid.

Mortality and Body Weight of Squab

Mortality of squabs in different groups during the experiment was calculated. Mortality in CON group was higher (P < 0.05) than that in MTZ, BAL, BAM, and BAH groups (Figure 1). The effects of adding bile acids in water on body weight were shown in Table 4, and there were no differences among these 5 groups at 28 d.

Figure 1.

Figure 1

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Mortality of squabs from 1 d to 28 d. Columns with different letters indicated significant difference (P < 0.05). Abbreviations: BAH, 0.125% bile acid; BAL, 0.05% bile acid; BAM, 0.075% bile acid; CON, control; MTZ, 0.05% metronidazole.

Table 4.

Effects of bile acid on body weight of squabs (g).

Groups1 1 d 3 d 7 d 14 d 21 d 28 d
CON 19.96 ± 2.17 54.54 ± 6.24a 146.32 ± 14.47b 299.41 ± 23.47 428.70 ± 36.61 460.34 ± 42.91
MTZ 20.79 ± 1.54 60.84 ± 9.67b 160.83 ± 17.92a 309.11 ± 25.62 437.92 ± 30.86 467.19 ± 32.71
BAL 21.01 ± 2.14 57.98 ± 7.28a 151.45 ± 14.97 b 309.70 ± 20.89 434.20 ± 30.77 461.52 ± 35.58
BAM 20.42 ± 1.70 57.36 ± 8.54a 152.81 ± 18.36b 305.14 ± 37.72 452.74 ± 29.56 485.09 ± 30.07
BAH 20.13 ± 1.55 57.87 ± 7.18a 144.84 ± 20.46b 307.95 ± 31.96 434.26 ± 32.42 466.93 ± 39.32
P value >0.05 <0.05 <0.05 >0.05 >0.05 >0.05
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a,b

Means within a column with different superscripts indicated significant difference (P < 0.05).

1

CON = control; MTZ = 0.05% metronidazole; BAL = 0.05% bile acid; BAM = 0.075% bile acid; BAH = 0.125% bile acid.

Carcass Characteristics, Organ Index, and Meat Quality

As shown in Table 5, carcass traits of squabs at 28 d from 5 groups were calculated carcass performances. Slaughter yield, empty yield, half-empty yield, breast muscle yield, and abdominal fat were not significantly influenced in metronidazole and bile acids treated group compared with CON group. Thigh muscle yield was higher in MTZ, BAL, and BAM groups than that in CON and BAH groups (P < 0.05). Organ index was shown in Table 6, liver index in MTZ group was significantly higher than that in BAL group (P < 0.05), and spleen index in MTZ group was significantly higher than that in CON, BAL, and BAH groups (P < 0.05). Other indexes had no significant differences among these 5 groups. As shown in Table 7, meat quality didn't have significant difference among these groups.

Table 5.

Effects of bile acid on carcass performance of squabs (%).

Groups1 Carcass yield Eviscerated yield Thigh muscle yield Breast muscle yield Abdominal fat yield
CON 89.61 ± 3.44 74.13 ± 1.31 7.69 ± 0.47b 24.80 ± 1.55 1.66 ± 0.76
MTZ 88.98 ± 2.46 71.87 ± 3.42 8.43 ± 0.49a 24.65 ± 2.39 1.45 ± 0.51
BAL 88.90 ± 1.64 75.01 ± 1.93 8.51 ± 0.58a 25.49 ± 1.52 1.55 ± 0.44
BAM 86.93 ± 0.98 73.85 ± 1.07 8.54 ± 0.89a 26.17 ± 1.39 1.58 ± 0.78
BAH 89.98 ± 3.41 73.91 ± 3.70 7.92 ± 0.40b 25.51 ± 1.94 1.60 ± 0.42
P value >0.05 >0.05 <0.05 >0.05 >0.05
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a,b

Means within a column with different superscripts indicated significant difference (P < 0.05).

1

CON = control; MTZ = 0.05% metronidazole; BAL = 0.05% bile acid; BAM = 0.075% bile acid; BAH = 0.125% bile acid.

Table 6.

Effects of bile acid on organ index of squabs (%).

Groups1 Cardiac index muscularis stomach index Gland stomach index Liver index Thymus index Bursa of Fabricius index Spleen index
CON 1.13 ± 0.12 2.22 ± 0.25 0.32 ± 0.07 2.87 ± 0.19ab 0.41 ± 0.26 0.19 ± 0.09 0.12 ± 0.05b
MTZ 1.16 ± 0.15 2.01 ± 0.15 0.33 ± 0.04 3.30 ± 0.51a 0.38 ± 0.14 0.17 ± 0.07 0.19 ± 0.06a
BAL 1.14 ± 0.19 2.12 ± 0.18 0.31 ± 0.04 2.83 ± 0.65b 0.48 ± 0.13 0.23 ± 0.11 0.13 ± 0.03b
BAM 1.24 ± 0.11 2.07 ± 0.43 0.32 ± 0.13 2.88 ± 0.29ab 0.51 ± 0.20 0.23 ± 0.12 0.16 ± 0.07ab
BAH 1.20 ± 0.20 1.78 ± 0.83 0.28 ± 0.13 2.92 ± 0.33ab 0.41 ± 0.17 0.20 ± 0.08 0.11 ± 0.02b
P value >0.05 >0.05 >0.05 <0.05 >0.05 >0.05 <0.05
Open in a new tab
a,b

Means within a column with different superscripts indicated significant difference (P < 0.05).

1

CON = control; MTZ = 0.05% metronidazole; BAL = 0.05% bile acid; BAM = 0.075% bile acid; BAH = 0.125% bile acid.

Table 7.

Effects of bile acid on meat quality of squabs.

Groups1 L a* b* pH12 pH23 Drip loss
CON 38.66 ± 1.33 20.38 ± 0.99 10.91 ± 1.44 6.00 ± 0.18 6.04 ± 0.12 5.62 ± 1.84
MTZ 40.60 ± 2.32 20.53 ± 0.37 11.61 ± 1.68 6.04 ± 0.14 6.05 ± 0.10 5.95 ± 2.23
BAL 39.17 ± 2.23 20.01 ± 0.46 11.40 ± 1.08 5.94 ± 0.16 5.92 ± 0.14 3.49 ± 3.12
BAM 40.07 ± 3.08 20.27 ± 1.05 11.12 ± 0.87 5.89 ± 0.18 5.93 ± 0.15 4.77 ± 2.90
BAH 39.45 ± 2.06 20.74 ± 0.92 10.65 ± 1.15 5.91 ± 0.13 5.93 ± 0.13 3.93 ± 2.55
P value >0.05 >0.05 >0.05 >0.05 >0.05 >0.05
Open in a new tab
1

CON = control; MTZ = 0.05% metronidazole; BAL = 0.05% bile acid; BAM = 0.075% bile acid; BAH = 0.125% bile acid.

2

pH1 1 h postmortem.

3

pH2 24 h postmortem.

Blood Metabolites

Serum biochemical metabolites of squabs were shown in Table 8. Concentrations of TC, HDL, LDL, AST, and IgG had no differences among these groups (P > 0.05). ALB in CON group was higher than that in other groups (P < 0.05). ALT in CON group was the highest in these groups, especially significant compared with MTZ, BAL, and BAH groups (P < 0.05). The level of sCD8 was significantly higher in MTZ and BAH groups compared with CON group (P < 0.05).

Table 8.

Effects of bile acid on serum biochemical index of squab in different groups1.

Items2 CON MTZ BAL BAM BAH P value
TC (mmol/L) 9.16 ± 1.06 8.37 ± 1.08 7.73 ± 0.30 8.16 ± 1.91 8.35 ± 0.71 >0.05
HDL (mmol/L) 6.57 ± 0.73 5.79 ± 0.72 5.72 ± 0.31 5.81 ± 1.34 6.12 ± 0.64 >0.05
LDL (mmol/L) 5.54 ± 0.82 5.27 ± 0.65 4.64 ± 0.08 5.06 ± 1.35 5.11 ± 0.65 >0.05
ALB (g/L) 29.36 ± 4.26a 15.80 ± 2.55b 16.87 ± 6.07b 15.88 ± 2.90b 17.61 ± 4.16b <0.05
ALT (U/L) 111.80 ± 9.68a 84.88 ± 12.21b 87.38 ± 6.51b 109.91 ± 6.27a 90.83 ± 14.42b <0.05
AST (U/L) 9.96 ± 0.38a 7.99 ± 0.80b 8.84 ± 0.50ab 9.02 ± 1.31ab 8.67 ± 0.62b >0.05
IgA (μg/mL) 164.87 ± 4.85ab 159.41 ± 2.70b 162.14 ± 3.42b 171.05 ± 8.22a 168.73 ± 7.39ab <0.05
IgG (μg/mL) 1566.57 ± 154.69 1570.28 ± 60.57 1568.39 ± 51.31 1746.07 ± 57.99 1680.45 ± 168.60 >0.05
sCD4 (U/mL) 7.57 ± 0.54a 7.18 ± 0.20a 6.36 ± 0.54b 7.87 ± 0.99a 7.76 ± 0.08a <0.05
sCD8 (U/mL) 99.14 ± 1.49b 105.18 ± 2.99a 103.02 ± 9.03b 108.77 ± 12.36ab 115.50 ± 8.12a <0.05
Open in a new tab
a,b

Means within a column with different superscripts indicated significant difference (P < 0.05).

1

CON = control; MTZ = 0.05% metronidazole; BAL = 0.05% bile acid; BAM = 0.075% bile acid; BAH = 0.125% bile acid.

2

HDL = high-density lipoprotein; LDL = low-density lipoprotein; ALB = albumin; ALT = alanine transaminase; AST = aspartate transaminase; IgA = immunoglobulin A; IgG = immunoglobulin G; sCD4 = soluble CD4; sCD8 = soluble CD8.

Histopathology

Mucosa of oral cavity, thymus, liver, and spleen was collected and stained with hematoxylin and eosin to observe the histopathology. The tissue lesions of CON group could be observed in Figure 2. Vacuolar deformation and disappearance of cell polarity occurred in basal layer cells of oral mucosa, and some cells in the acanthus layer showed insufficient keratinization. Inflammatory cells infiltrated in thymus, liver, and spleen tissues. Hemorrhage in cortical area and partial tissue necrosis were observed in thymus.

Figure 2.

Figure 2

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Observation of tissues of squabs in hematoxylin and eosin staining. Abbreviations: BAH, 0.125% bile acid; BAL, 0.05% bile acid; BAM, 0.075% bile acid; CON, control; MTZ, 0.05% metronidazole.

Duodenal Histological Analysis and Microbial Diversity

Duodenal VH, CD, and VH/CD ratio were shown in Table 9. Drinking supplement of bile acids had no significant effect on VH, but it could increase CD in BAH group compared with CON group (P < 0.05). VH/CD ratio in BAH group was significantly lower than that in CON group (P < 0.05), but there were no significant differences among CON, MTZ, BAL, and BAM (P > 0.05).

Table 9.

Intestinal morphology of the duodenum of squab.

Groups1 Villus height Crypt depth VH/CD ratio
CON 477.69 ± 76.85a 84.37 ± 15.96a 5.79 ± 1.21ac
MTZ 421.11 ± 89.43b 91.35 ± 12.41a 4.65 ± 0.99abc
BAL 516.05 ± 100.63a 86.00 ± 17.60a 6.25 ± 1.97a
BAM 505.35 ± 26.39a 90.09 ± 30.64a 6.12 ± 1.77a
BAH 501.96 ± 34.92a 121.22 ± 17.20b 4.22 ± 0.70b
P value <0.05 <0.05 <0.05
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a–c

Means within a column with different superscripts indicated significant difference (P < 0.05).

1

CON = control; MTZ = 0.05% metronidazole; BAL = 0.05% bile acid; BAM = 0.075% bile acid; BAH = 0.125% bile acid.

Colon Microbial Diversity and Composition

The 16S rRNA gene sequencing was performed to investigate the colon microbiota of squabs from CON, MTZ, and BAH groups. After size filtering, quality control, and chimera removal, the effective reads of CON, MTZ, and BAH groups were 388,002, 390,335, and 368,486, respectively, then they were decomposed into 571, 477, and 539 operational taxonomic units (OTUs) based on the 97% identity level. Venn diagram showed the common OTUs of the 3 groups were 447 (Figure 3). The abundance-based coverage estimator (ACE) and Simpson index were counted to reflect the alpha diversity. ACE index in BAH group was significantly higher than that in CON and MTZ groups, and it was higher in CON group compared with MTZ group (P < 0.05, Figure 4A). Simpson index in CON and BAH groups was higher than MTZ group (P < 0.05, Figure 4B). Beta diversity was illustrated by principal coordinate analysis (PCoA) in Figure 5, which showed remarkable differentiation of the microbial community among the 3 groups. The CON group and MTZ group were separated, whereas the group treated with BAH was included in the CON group. The relative abundance at phylum and genus levels was studied and shown in Figure 6. At the phylum level, Firmicutes and Proteobacteria were the major bacteria in the colon of squabs, accounting for more than 93% of the total colon bacterial community (Figure 6A). In Figure 6B, the top 11 genera were listed. The relative abundance of Lactobacillus was the highest among these bacteria, that were 77.21%, 91.20%, and 73.19% in CON, MTZ, and BAH, respectively.

Figure 3.

Figure 3

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Number of intestinal microbe OTU of squab in each group. Abbreviations: BAH, 0.125% bile acid; CON, control; MTZ, 0.05% metronidazole.

Figure 4.

Figure 4

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The abundance-based coverage estimator (ACE) and Simpson index in colon. Columns with different letters indicated significant difference (P < 0.05). Abbreviations: BAH, 0.125% bile acid; CON, control; MTZ, 0.05% metronidazole.

Figure 5.

Figure 5

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Principal component analysis of microbial structure in colon. Abbreviations: BAH, 0.125% bile acid; CON, control; MTZ, 0.05% metronidazole.

Figure 6.

Figure 6

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Species abundance of colonic contents at phylum level (A) and at genus level (B). Abbreviations: BAH, 0.125% bile acid; CON, control; MTZ, 0.05% metronidazole.

DISCUSSION

The protozoan T. gallinae, a flagellated parasite of oropharynx, can lead to avian disease trichomonosis. The main host of T. gallinae is columbidae, including the domestic and wild pigeon, which has been considered to be responsible for the worldwide spread of T. gallinae and causes enormous economic loss (Harmon et al., 1987). Birds infected by T. gallinae can cause necrotic lesions not only in the upper digestive tract as oropharynx and crop, but also the organs as liver, air sacs, and cranium (Marx et al., 2017). In our results, squabs in CON group affected by T. gallinae could see serious pathological lesions in oral cavity, thymus, liver, and spleen. Borji et al. (2011) also reported the affected pigeon appeared heavy infiltration of inflammatory cells in the tracheal mucosa, granulomatous lesion in the liver and tubulointerstitial nephritis in the kidney (Borji et al., 2011). T. gallinae transmits by contaminated feed and water, and can also occur from parent pigeons to their squabs by crop milk (Krone et al., 2005). In our results, the number of oral T. gallinae in squabs was associated with that in parent pigeons.

Nitroimidazoles especially metronidazole are always used as drugs to treat trichomonosis. However, the increasing number of clinical resistant strains and the policy of banning to use antibiotics to prevent disease in animal husbandry have encouraged the development of new therapeutic strategies (Zimre-Grabensteiner et al., 2011; Rouffaer et al., 2014; Tabari et al., 2021; Bailén et al., 2022). Bile acid species as a natural product has been reported participating in host immunity as signaling molecules (Fiorucci et al., 2018). Bile salt was also reported have the ability to display antigiardia lactobacilli effect. Allain et al. (2018) reported bile-salt hydrolase activity was able to mediate antigiardial effect by generating deconjugated bile salts, which could reduce the trophozoites load of the parasite in small intestine of mice (Allain et al., 2018). While the effects of bile acid or bile salt on preventing and treating trichomonosis have neither been reported in vivo nor in vitro. In our studies of parent pigeons, the numbers of T. gallinae didn't have significant difference in the beginning. After different treatment, number of T. gallinae in the CON group increased continually and significantly higher than other groups during all the experiment. Groups treated with metronidazole, 0.075 and 0.125% bile acids could inhibit T. gallinae growth effectively. The numbers of oral T. gallinae in squabs had the same trend as the parent pigeons in 5 groups. Meanwhile, mortality of squabs in CON group was significantly higher than other groups and obvious tissue lesions was observed. Groups treated with metronidazole and 0.125% bile acids had the lower mortality. These results were consistence with previous report that trichomoniasis could cause pigeon death by lesions in organs (Chavatte et al., 2019; Martínez-Herrero et al., 2020; Brunthaler et al., 2022).

The bile acids are powerful regulators of the metabolism. They could promote intestinal absorption of lipids through the enterohepatic circulation, increase water solubility of nonpolar lipids as cholesterol promoting their delivery to intestinal epithelium, and activate specific nuclear receptors and G-protein coupled receptors (Russell, 2009; Šarenac and Mikov, 2018). So exogenous bile acids were supplemented in feed or drinking water sometimes to improve growth performance. Peng et al. (2019) have found 240 mg/kg bile acids supplementation effectively enhanced growth performance and enhanced intestinal immune function in grass carp (Peng et al., 2019). In chicken, dietary supplementation with 60 and 80 mg/kg of bile acid could significantly improve growth performance and carcass traits (Lai et al., 2018). While in our results, body weight, carcass traits, and meat quality didn't have significant differences among treatments. Body weights of pigeons were unaffected by drinking supplementation with bile acids that might be because the concentration of bile acids used in our reports was lower for the purpose of our study was to inhibit trichomoniasis. Thymus, spleen, and bursa of Fabricius are the main immune organs of birds (Khalaifa, 2014). We calculated organ indexes of these organs. Spleen index of MTZ group was significantly higher than CON and BAH group. Liver index of MTZ was also high in these treatments and higher than that of BAL group significantly in squabs. Metronidazole has adverse effects such as nausea, abdominal pain, diarrhea, neurotoxicity, and cancer (Hernández et al., 2019), and they may be delivered from parent pigeons to squabs by crop milk. Liver is the main metabolic organs of metronidazole, so this may result in higher liver index in MTZ group (Lamp et al., 1999).

Serum levels of alanine (ALT) and aspartate (AST) aminotransferases are the markers of liver injury (Sookoian and Pirola, 2015), which are the consequence of the liver cell membrane damage with the subsequent leakage of intracellular enzymes (Kamiike et al., 1989). In our results, ALT and AST were higher in CON groups than in metronidazole and bile acids treated groups, indicating that trichomoniasis could induce liver damage and both metronidazole and bile acids could decrease this damage. Bile acids are regulators not only in metabolism promoting nutrients absorption, but also in immunity regulating immune system activation (Fiorucci et al., 2018). Soluble CD8 antigen serum levels of bile acids treated groups were higher compared to CON group in our results. Soluble CD4 and CD8 are markers of immunological activation and enable T cell subset activation (Ninova et al., 1994). Our results indicated that squabs in bile acids treated group could be activated adaptive immunity more efficiently than other groups.

The major function of intestine is digestion and absorption of nutrients by intestinal mucosa, especially the villi (Wang et al., 2019; Xie et al., 2020). The villus height, crypt depth, and VH/CD ratio are closely related to the intestinal function (Miśta et al., 2017; Peebles et al., 2019). The intestine, especially the villi, is the major site of nutrient absorption (Wang et al., 2019). The villus lengths and crypt depths have been considered to determine the ability of nutrient absorption of villi (Miśta et al., 2017; Peebles et al., 2019). Increased villus height, shallow crypt depth, and high VCR indicated enhanced digestion and absorption of the intestine (Pirarat et al., 2011). In this study, duodenal villus height in BAL group was significantly higher than other groups. VH/CD ratio in BAL group was high but not significant. This study confirmed that bile acid improved intestinal morphology, indicating that drinking supplementation of bile acid was beneficial to intestinal health. Hagio et al. (2015) also reported bile acids could promote proliferation and survival of the intestinal epithelium (Hagio et al., 2015).

Primary bile acids are synthesized in liver and secreted into colon during the enterohepatic circulation (Allegretti et al., 2016). The gut microbial community metabolizes primary to secondary bile acids, which are capable of regulating their own synthesis, glucose and lipid homeostasis, intestinal microbial composition, and serve as therapeutic agents (Ridlon and Bajaj, 2015). The metabolism of primary to secondary bile acid could be disrupted by antibiotics initiating some metabolic diseases (Allegretti et al., 2016). This experiment showed the hitherto unexplored role of bile acids and metronidazole controlled the colonic microorganisms’ population of squabs by adding these materials in drinking water of parent pigeons. In this study, the alpha diversity index of bile acid treated group and CON group were significantly higher than that of metronidazole treated group. Higher diversity of intestinal microorganisms indicates better maintenance of the dynamic balance of the microecosystem and intestinal health (Bortoluzzi et al., 2017; Sun et al., 2022). Islam et al. (2011) have showed that bile acid had bactericidal activity for its hydrophobicity, which increased its affinity of the bacterial cell membrane (Kurdi et al., 2006; Islam et al., 2011). These results indicated that drinking supplementation with bile acid could be beneficial to the balance of the intestinal health. This study demonstrated significant differences among CON, MTZ, and bile acid groups of colonic microbiota phylum in the abundance of Firmicutes and Proteobacteria. Bacteria in Firmicutes can promote hydrolysis and utilization of carbohydrates (El et al., 2013; Zhang et al., 2022), so they have advantage in utilization of nutrients and maintenance of body health (Videnska et al., 2014).

In conclusion, drinking water supplemented of 0.05 to 0.125% bile acid in this study could efficiently inhibit growth of T. gallinae in both parent pigeons and squabs. Squabs infected with T. gallinae in control group had higher mortality rate and more serious tissue lesions especially liver injury than squabs in other groups. Squabs in bile acids treated group could be activated adaptive immunity more efficiently than other groups and they were improved intestinal morphology and health by bile acids.

Acknowledgments

ACKNOWLEDGMENTS

This study was funded by Beijing Innovation Consortium of Agriculture Research System (BAIC06-2022); Agricultural Science and Technology Innovation Program (ASTIPIAS04). The authors would like to thank all members of this work for their advice and technical.

DISCLOSURES

The authors declare that they have no conflicts of interest to this work.

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