Effect of dietary oridonin supplementation on growth performance, gut health, and immune response of broilers infected with Salmonella pullorum

Background The effects of dietary supplementation of oridonin (ORI) on growth performance, cecal microbiota, epithelium development and antioxidant and immune parameters of broilers infected with S. pullorum were studied. A total of 300 1-d-old male chicks were selected and divided into 5 trial groups (6 replicates of 10 chickens): 1) nonchallenge control chicks (CON), 2) chicks treated with Salmonella Challenged Control (SCC), 3) chicks treated with S. pullorum challenge and 50 mg/kg ORI (O1), 4) chicks treated with S. pullorum challenge and 80 mg/kg ORI (O2), and 5) chicks treated with S. pullorum challenge and 100 mg/kg ORI (O3). Results The results showed that S. pullorum had no effect on the feed intake (FI), body weight gain (BWG) or feed conversion ratio (FCR) of broilers compared with the values measured for the CON group (P > 0.05). However, compared with the characteristics of CON, S. pullorum showed effects on the counts of Salmonella and Lactobacillus at 7 d and at 14 d (P < 0.05), on jejunal development at 7 d (P < 0.05), and on jejunal immunoglobulin A (IgA) concentration at 7 d (P < 0.05). The addition of 100 mg/kg ORI had the greatest effect on the counts of Lactobacillus and Salmonella in cecal content (P < 0.05), malonaldehyde (MDA) content in the jejunum (P < 0.05), villi height of the small intestine, and IgA concentrations in the jejunum (P < 0.05). Conclusions The results suggest that ORI can improve Salmonella-induced immune responses and protect intestinal health, not only through its immune inhibitory properties but also through its multi-protective effects on gut health.


Background
S. pullorum is a capnophilic gram-negative rod-shaped bacterium that can cause pullorum disease (PD). Pullorum disease was once enzootic in many areas of the world [1]. Broiler chickens are known to be extremely sensitive to S. pullorum infections in the first 7 d of their life because of delayed development and establishment of their normal intestinal flora. S. pullorum infection can cause nutritional (protein, amino acid, and vitamin) deficiency, intestinal flora disorder, reduction in production performance, and invasion of internal organs in young birds, resulting in significant economic losses to the poultry industry [2]. Therefore, several synthetic chemicals (such as zinc-bearing clinoptilolite) have been used to control or limit the intestinal colonization and invasion of S. pullorum in poultry production [3].
Isodon rubescens (Donglingcao in Chinese) is widely used in traditional Chinese medicine and has long served as a popular medicine for respiratory and gastrointestinal bacterial infections, inflammation, and cancer [4]. Oridonin (ORI) is claimed to be a valid natural compound and one of the richest ent-kaurane diterpenoids of Isodon rubescens. ORI has generated much interest because of its notable pharmacological and biological activities, such as its anti-bacterial and anti-inflammatory properties and its ability to control viral replication, eliminate active oxygen radicals, and protect other internal organs, among other abilities [5]. Substantial research efforts have shown that isolated oridonin is used alone or in combination with other drugs to prevent and cure bacterial infection in vivo and in vitro, and since 1976, oridonin has been known to exhibit antimicrobial activity against gram-positive bacteria [6]. However, to the best of our knowledge, few studies have focused on the effects of dietary supplementation of oridonin or derivatives on antimicrobial activity against S. pullorum in animals, with no study addressing broiler chickens. Hence, the aims of this work were to evaluate the beneficial effects of oridonin supplementation on the growth performance, intestinal microbiota, and gut morphology of broiler chickens and to validate whether dietary oridonin supplementation could attenuate damage to the intestinal lining and protect broilers from negative effects of S. pullorum.

Bird husbandry and experimental diets
Three hundred 1-d-old male Arbor Acres (AA) broilers were obtained and divided into five dietary treatments, each with six replicates, 10 chickens per replicate, for a 21-d feeding trial. The 5 treatments were as follows: 1) nonchallenge control chicks (CON), 2) chicks treated with Salmonella Challenged Control (SCC), 3) chicks treated with S. pullorum challenge and 50 mg/kg ORI (O1), 4) chicks treated with S. pullorum challenge and 80 mg/kg ORI (O2), and 5) chicks treated with S. pullorum challenge and 100 mg/kg ORI (O3). All birds were raised in multi-tiered brooder cages and kept in an environmentally controlled room. During the study period, birds had ad libitum access to water and a balanced unmedicated diet meeting or exceeding the recommendations of the National Research Council (NRC, 1994). The basal (starter) diets were based on corn and soybean meal, as shown in Table 1, and provided as a mash. The experimental design and procedures were approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University.
ORI used in the experiment was purchased from the Laieryin Biological Technology Company Limited (Luoyang, Henan province, P. R. China) with a purity of 98%.

Salmonella infection model
The strain of S. pullorum (CVCC 533) was obtained from the China Veterinary Culture Collection Center (Beijing, P. R. China). Broilers in the SCC, O1, O2, and O3 experimental groups were orally treated with 4 × 10 4 CFU S. pullorum bacteria per bird on d 3 posthatch, and the chicks of the CON group were treated with an equal volume of physiological saline.

Sample collection and procedures
Four, 11, and 18 days after S. pullorum infection, one chick per replicate was selected at random, weighed after a 12-h feed restriction, and sent to the Veterinary Laboratory for a bacterial culture of the cecal contents. Subsequently, the broilers were euthanized by cervical dislocation, and an approximately 2-cm-long section of the proximal jejunum was extracted, washed, and fixed for histological examination. The proximal jejunal mucosa was excised carefully, frozen, and kept for further analysis.

Growth performance
Body weight was measured at 1, 7, 14, and 21 d of age. The amounts of feed supplied and feed waste were also weighed to calculate the feed intake (FI) and feed conversion ratio (FCR). Mortalities were recorded daily to calibrate growth performance parameters.

Intestinal histomorphology
Three jejunum cross-sections were prepared using standard paraffin embedding procedures by sectioning at a thickness of 5 μm and staining with hematoxylin and eosin. The jejunum villus height and crypt depth of the stained sections were examined with an Axioplan-2 optical microscope (Carl Zeiss Jena GmbH, Jena, Germany) coupled with a refrigerated QImaging Retiga 4000R digital camera (QImaging, Surrey, British Columbia, Canada) with a charge-coupled device detector, and were expressed in micrometers (μm). A total of 15 complete, well-oriented crypt-villus units were measured with an image processing and analysis system (Version 1, Leica Imaging Systems Co., Ltd., Cambridge, UK) for each type of tissue from each broiler.

Mucosal antioxidant and immunity index
Approximately 0.3 g of intestinal mucosa samples was homogenized and centrifuged. The supernatant was used for examining mucosal antioxidant and immunity indices. Total superoxide dismutase (T-SOD) activity and malonaldehyde (MDA) content were measured using diagnostic kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, P.R. China). The levels of immunoglobulin G (IgG) were examined using a commercially available 125 I radioimmunoassay kit with goat anti-chicken IgG (BlueGene, Shanghai, People's Republic of China). The assay was responsive to a test limit of 0.1 g/mL. The concentration of intestinal immunoglobulin A (IgA, BlueGene, Shanghai, People's Republic of China) was determined based on the method described by a chicken IgA enzyme-linked immunosorbent assay.

Statistical analysis
An analysis of variance was performed using the General Linear Model procedure of the Statistical Package for Social Sciences 20.0 (SPSS Inc., Chicago, IL, USA) in a completely randomized design. The differences among all the treatment means were identified using the Tukey's range test at levels of significance P ≤ 0.05.

Growth performance and microbial population
Neither S. pullorum infection nor ORI supplementation had an effect on FI, BWG, FCR or mortality (Table 2). Throughout the evolution of the infection, Salmonella reduced the population of Lactobacillus at 7 and 14 d and promoted the growth of Salmonella colonies in challenged chickens compared with that of the control group (P < 0.05) ( Table 3). However, ORI supplementation increased the counts of Lactobacillus (with the exception of O1 and O2, at 21 d) and reduced the Salmonella population (excluding the O1 group, at 21 d) (P < 0.05) compared with that of the non-supplemented SCC group.

Intestinal histomorphology
The SCC group challenged by S. pullorum showed a decreased jejunal villus height and V/C value (P < 0.05) and an increased jejunal crypt depth at 7 d (P < 0.05) compared with those of the CON group (Table 4). ORI supplementation increased the villus height of chickens at all inclusion levels, increased V/C, and decreased crypt depth at 100 mg/kg compared with the values measured for the SCC group (P < 0.05). However, the

Antioxidant index
The MDA content in SCC was increased at 21 d (P < 0.05) compared with that of CON (Table 5). Moreover, the addition of ORI reduced the MDA content in the jejunal mucosa at 21 d (P < 0.05) compared with that of the SCC group. Compared with that of the SCC group, the MDA content in the jejunal mucosa of 7-d-old chicks did not decrease in the O1 or O2 group (P > 0.05), but it did decrease in the O3 group (P < 0.05). However, there were no differences in MDA content in the jejuna mucosa at 7 or 14 d. The T-SOD activity in jejunal mucosa at 7 d or 21 d did not differ among groups (P > 0.05). The T-SOD activity in the SCC group decreased at 14 d (P < 0.05) compared with that of CON (P < 0.05). The incorporation of 100 mg/kg ORI increased T-SOD activity at 14 d.

IgA and IgG concentration
Salmonella infection increased the concentrations of jejunal IgA and IgG at 7, 14, and 21 d (P < 0.05) ( Table 6). Dietary ORI decreased jejunum IgA and IgG contents at 7, 14, and 21 d (P < 0.05) compared with those of the SCC (P < 0.05). However, there were no differences in the concentration of jejunal IgA or IgG between the CON and ORI group at 7, 14 or 21 d (P > 0.05).

Discussion
Salmonella infections resulting in slowed production performance, intestinal colonization, inflammation responses, and invasion of internal organs in broiler chickens have been reported [7]. In the present study, Salmonella infection had no effect on the production performance of the chicks, but certain short-term symptoms, such as reluctance to move, emotional panic, lethargy, lack of appetite, and decreased water intake   Values within a row not sharing the same superscript are different at P < 0.05; n = 6 Table 5 Effect of oridonin on the jejunal mucosa antioxidation function of broilers challenged with S. pullorum Values within a row not sharing the same superscript are different at P < 0.05; n = 6 appeared at 6 d of age, after the first inoculation. There were no apparent clinical symptoms at 11 or 18 d. This finding is consistent with that of Chen et al. (2015), who noted that clinical symptoms vanished after operative treatment [8]. However, in some studies, the effects of salmonella were more severe likely due to age, bacterial serotype, dose or environmental conditions [3]. In any case, ORI did not show any effects on productive performance likely because of the lack of an effect of infection on such variables.
In previous studies, Salmonella caused a high level of infection in the caecum [9] and decreased the number of Lactobacillus colonies [10]. In the present study, the Salmonella count in SCC groups was higher than that of uninfected groups, whereas Salmonella inhibited the growth of Lactobacillus. Furthermore, Lactobacillus numbers markedly increased and Salmonella numbers decreased in the ORI treatment, with values similar to those of the control group. These results indicate that ORI can counteract Salmonella infection, promoting the growth of Lactobacilli to levels comparable to those in healthy chickens [11].
Villus height and crypt depth are important indices of the functional capacity of the enterocytes of broilers, and the VH:CD ratio has been shown to immediately affect the digestion and absorption of the intestinal mucosa [12]. In the current study, nearly all measured morphometric parameters of the jejunum were affected after 4 d post infection. This finding agrees with the observation that Salmonella leads to apoptosis of cells in the jejunum [13]. Such processes tend to be attributed to the impairment of epithelial protein synthesis and function after Salmonella infection, which dramatically influence the early intestinal morphological development of chickens. As previously noted, dietary inclusion of ORI resulted in increased villus height and VH:CD ratio and reduced crypt depth in broilers. The development of jejunal villi may be enhanced by the antibacterial and anti-inflammation properties of ORI.
Some studies have shown that intestinal oxidative stress and substantially impaired mucosal barriers occur in Salmonella challenged broilers [8]. The results of this study showed that Salmonella infection led to an imbalance of cytosolic redox status in favor of prooxidants, causing intestine cells to experience a state of oxidative stress in infected broilers. The results demonstrate that intestinal mucosal barriers were substantially disrupted in Salmonella challenged broilers. The molecular mechanisms of this mucosal barrier injury are completely unknown but may be due to the strong oxidative burst against Salmonella infection. However, administration of ORI to infected animals has maintained cellular antioxidant defense systems (T-SOD) at their normal levels, which might be due to a reduction in oxidant lesions by regulating the oxidation of T-SOD enzymes in the intestinal immune response.
The small intestine is one of the most important parts of the mucosal immunity system and provides antigen-specific protection by producing antibodies. Certain bacteria and viruses affect immunoglobulin synthesis, and Salmonella could induce marked intestinal immune responses characterized by the secretion of a large number of immunoglobulins in animals [14,15]. Thus, we investigated the effects of Salmonella challenge on immunoglobulins in the jejunum of broilers. The results verified that SCC chicks experienced increased concentrations of jejunal IgA and IgG in response to Salmonella intestinal infection.
IgA and IgG, the major immunoglobulins produced by lymphocytes of the mucosa, are clearly involved in the development of the intestinal immune response to Salmonella and are critical for protecting mucosal surfaces against toxins, viruses, and bacteria by neutralizing them or preventing them from binding to the mucosal surface [16][17][18]. In the present study, we found that ORI can decrease immunoglobulin concentrations (IgA and IgG) in the jejunum. The mechanisms of ORI's immune-protecting properties may be partially attributed to T cell depletion in the peripheral immune system [19]. Additional studies will be required before the potential applications of ORI in feeding practices are fully understood.

Conclusions
In conclusion, our results suggest that S. pullorum can reduce the counts of Lactobacillus and the villi height of the small intestine and increase the counts of Salmonella, IgA concentrations in the jejunum, and the MDA content in the jejunum. ORI could protect the Values within a row not sharing the same superscript are different at P < 0.05; n = 6 intestinal epithelium and normalize bacterial populations and immune response in Salmonella challenged broilers.