Preventive activities of Scutellariae Radix, Gardeniae Fructus, and probiotics in Salmonella enterica serovar Typhimurium infection in chickens

Highlights

• Effects of dietary supplements with SR, GF, and LAB in infected chickens.

• Herbs and LAB decreased Salmonella load in feces and intestinal tissues.

• Levels of IFN-γ and IL-1β in intestinal tissues were reduced in all treated groups.

• SR and GF with LAB suppressed the levels of IL-8 in serum and intestines.

• Herbs and LAB could be a natural way to replace antibiotics in feed additives.

Abstract

In this study, Scutellariae radix (SR), Gardeniae fructus (GF), and probiotic-lactic acid bacteria (LAB) were evaluated to determine their efficiency in preventing an infection from Salmonella enterica serovar Typhimurium in chickens. Chickens were fed with a diet supplied with either LAB (0.5% of diet), SR (0.5% of diet), SR with LAB (0.5% of diet each), GF (0.5% of diet), GF with probiotics (0.5% of diet each), or a basal diet as a control for 10 days before being exposed to a 10¹⁰ CFU dose of ST315. Clinical signs were monitored for 6 days post-challenge. The minimal dosage of S. Typhimurium strain ST315 used to infect chickens without causing serious sickness is 10¹⁰ CFU/ml. The maximum amount of bacteria that was shed in feces was achieved 3 days after the initial infection. Herbs and LAB supplements reduced Salmonella fecal shedding and the bacterial load in intestinal tissues (P < 0.05). In all treatment groups, the mRNA levels of IFN-γ and IL-1β in intestinal tissues were decreased (P < 0.05). Furthermore, the SR group and GF with LAB group also saw suppression of the serum and intestinal levels of IL-8 (P < 0.05). Our findings suggest that herbs and probiotic-LAB can clear the bacterial infection as well as suppress the inflammation response. The combination of herbs and LAB also has potential in the prevention of an infection when supplied as feed additives.

Introduction

Livestock have a higher sensitivity toward infectious diseases, such as salmonellosis, because they are fed in high density farms (Minga et al., 1985, Odontsetseg et al., 2005, Davies et al., 2013). Salmonella species—rod-shaped, aerobic, Gram-negative, flagellated bacteria—are causative agents of salmonellosis in humans and can cause severe infections in animals (Mead et al., 1999). There are estimated to be over 10 million cases of Salmonella infection reported all over the world each year (Pang et al., 1995); thus it is a major public health problem and creates a severe economic impact in many countries. Until 2004, over 2,500 Salmonella serotypes were identified (Popoff et al., 2004). Among them is S. enterica serovar Typhimurium, a crucial serotype responsible for a food-borne disease transmitted from animals to humans (Mead et al., 1999). After the host is infected by S. Typhimurium, gastroenteritis is triggered and usually self-limited (Darwin and Miller, 1999). Bacteria enter into the intestinal epithelial cells and disturb host cells’ membranes (Francis et al., 1992, Francis et al., 1993), as well as invade macrophages and induce apoptosis (Monack et al., 1996).

In order to prevent a Salmonella infection or treat infected animals, many antibiotics are used as feed supplements (Cromwell, 2002, Jung et al., 2010). Use of antibiotics in feed not only leads to animal products such as meat, eggs, and milk containing residual amounts of the antibiotics (Nisha, 2008, Yang et al., 2009, Namagirilakshmi et al., 2010), but it also elevates the development and spread of antibiotic-resistant Salmonella strains, which in turn causes severe public health problems (Gross et al., 1998, Angulo et al., 2000, Chiu et al., 2002, Tseng et al., 2014). Therefore, there is an emergent need to find safe, efficient, and natural feed supplements to decrease the overuse of and overreliance on antibiotics. One of the most popular methods in alleviating antibiotic abuse is using a preventative treatment with natural immune-stimulants—like probiotics and herbs—to enhance the immune system of livestock against microbial infections (Harikrishnan et al., 2009, Janardhana et al., 2009, Jung et al., 2010, Liu et al., 2010)

Some medicinal herbs have been used in managing the health of domesticated animals since ancient times (Eevuri and Ramya, 2013, Khosravifar et al., 2014), such as Scutellariae radix (SR) and Gardeniae fructus (GF) (Katsuyama, 1994, Chang et al., 2013). Therefore, replacing in-feed antibiotics with natural supplements, such as the aforementioned medicinal herbs, should be regarded as a good candidate and substitute. In previous studies, Chinese herbal medicines have been used in prophylactic administration or treatment against Salmonella infection (Lee et al., 2006, Kim et al., 2008, Kwon et al., 2008, Chang et al., 2013). Unlike antibiotics, herbal compounds can be absorbed quickly in the intestine (Khosravifar et al., 2014). In addition, their half-lives are shorter, so the accumulation of these compounds are less likely to be found in the treated animal’s tissues and therefore there will be minimal residue in the animal products (Kohlert et al., 2000).

Another natural immunostimulant, probiotics, has been studied extensively for many years and used as an in-feed supplement (Lin et al., 2007, Tsai et al., 2011, Chang et al., 2013). Probiotics are microorganisms that are claimed to provide a health benefit to the host when consumed in adequate amounts (FAO/WHO, 2001, Borchers et al., 2009). The microbial species most commonly used in probiotic application are the lactic acid bacteria (LAB) strains, especially Lactobacillus spp. Sufficient evidence shows LAB strains enhance host immunity and protect the host against infection through gastrointestinal pathogens (Pascual et al., 1999, Mercenier et al., 2003, Lin et al., 2007, Castillo et al., 2011, Tsai et al., 2011). They can also act as immunomodulators or adjust the composition and activity of intestinal microbes (Naidu et al., 1999). Therefore, LAB strains have the potential to improve the host’s intestinal microbial balance. Moreover, intestinal microflora can also play a crucial role as probiotics (Lievin-Le Moal and Servin, 2006) and transform herbal components into bioactive compounds, e.g., converting ginsenoside Rb1 to compound K, which is more active and subsequently absorbed (Wakabayashi et al., 1998, Lee et al., 1999, Bae et al., 2002, Yim et al., 2004).

In a previous study (Chang et al., 2013), we screened some medicinal herbs with a mouse infection model for potential preventive agents. We found that SR and GF could eliminate S. Choleraesuis in the spleen and intestine of infected mice. In a swine infected model, with exposing said swine to SR and GF before infection, SR and GF were found to have high antimicrobial activity and reduce TNF-α levels in the serum induced by S. Choleraesuis in the infected swine. Also, we found that LAB strains exhibited interesting properties in the swine infection model, such as preventing an infection, immune system enhancement, and adjusting the enzyme activity of intestinal microorganisms toward bioconverting compounds from medicinal herbs.

Besides pigs, chickens are also important livestock animals in many countries. In recent years, chickens have been found to be suffering from diarrhea and anemia caused by Salmonella infection as they grow (Baumler et al., 2000, Shivaprasad, 2000). This results in a serious problem for many countries’ poultry industries (Lee et al., 2007). In this study, we set out to determine whether SR and GF could be effective in preventing chickens from being infected by the field-collected and multi-drug resistant S. Typhimurium strain. The effects of LAB strains mixed with SR or GF as chicken feed supplements against Salmonella infection were also studied. Applying a mixture of LAB strains and medicinal herbs as feed additives, then, could be considered as an option for improving immunity and digestive abilities of chickens and perhaps other livestock.