Effect of feeding Chinese herb medicine ageratum-liquid on intestinal bacterial translocations induced by H9N2 AIV in mice

Avian influenza virus subtype H9N2 (H9N2 AIV) is usually found in chicken, but also isolated from the mammals, even humans [1, 2]. Importantly, most high pathogenic AIV (e.g. H7N9) acquired their internal’ gene segments from H9N2 strains [3]. And the virus can be isolated from host tissues including trachea, lung, brain, spleen, pancreas, cloacal cavity and intestinal tract. However, no characteristic clinical symptoms and lesions were observed in animals individually infected with H9N2 AIV. Generally, hosts begin to develop inflammation and enteric problems at 3–5 days post infection, often resulting in morbidity or mortality due to infection with secondary bacteria, for example Escherichia coli [4]. In mammals, AIV H9N2 always causes mild respiratory illness, but fatal outcomes are sometimes observed [3, 5, 6]. After H9N2 AIV infection, the inflammation was serious, even with severe peritonitis, perihepatitis and pericarditis in chicken. The problem of bacterial secondary infection has also plagued many scholars and clinical practitioners, and also brought great difficulties to the prevention and control of the bird flu. This is a puzzle that animal infection with influenza virus would appear serious bacterial secondary infection and how do these bacteria break through the body’s mucosal barrier into vital organs? All of those questions deserve further research.

The intestinal tract is the body’s largest immune organ. Once the mucosal barrier and microbial flora are destroyed, the intestinal diseases will occur. The avian influenza virus can be replicated in intestinal epithelial cells after infection and will trigger the massive expression of inflammatory factors and appear intestinal inflammatory injury [7, 8]. In clinically, antibiotics were always used to treat. Antibiotics can kill bacteria, but can also cause damage to intestinal beneficial flora, aggravating intestinal injury because of its broad spectrum. A large number of studies have showed that Chinese herbal medicine in the regulation of the intestinal tract have a good effect, such as Ageratum-liquid (AL), which can regulate gastrointestinal motility function, repair damaged mucosa and reduce intestinal permeability [9, 10]. As a representative natural medicine, AL is considered safe and with few adverse drug reactions. Clinical studies found that AL has significant effect on the treatment of gastrointestinal influenza [11, 12]. In the view of these problems, this project, on the basis of previous studies, we used BALB/c mice as an animal model to infect H9N2 AIV with low pathogenicity to investigate the mechanism of H9N2 AIV promoting bacterial translocation in the intestine of mice infected with H9N2 AIV, the close relationship between intestinal bacterial translocation and secondary bacterial infection, and the feasibility of compound traditional Chinese medicine in preventing intestinal flora translocation to prevent secondary infection.

H9N2 AIV, a low-virulent virus, is widely found in some avian species, and mammals, causing significant losses due to diarrhea and secondary infections [1, 2, 15]. There are many studies reporting the effects of H9N2 AIV on the respiratory tract [16, 17], but few reports focus on the effect of H9N2 AVI on the gastrointestinal tract. Stable intestinal morphology and physiological function are the basis of the intestinal health. It has been reported that short villus height and deep crypt depth means the poor intestinal function. The higher to the V/C value, the healthier to the intestine [18, 19]. In this study, the villus of ileum became shorter, the crypt became deeper, and V/C was down-regulated significantly after 5 and 12 dpi, indicating that mice infected with H9N2 AIV damaged the structure of villus and affected the function of nutritional absorption. The change of villi epithelial cells could be directly observed through pathological sections, showing, in accordance with previous observations [7, 20], H9N2 AIV infection could destroy the intestinal barrier structure, weaken digestion and absorption capacity, and further may affect the body’s immune capacity, resulting in that conditional pathogens might reach to pathogenic conditions.

It has been reported that H9N2 AIV infection could change the structure of intestinal flora with beneficial bacteria reduction and damage the structure of intestinal barrier in mice or chicken [7, 20, 21]. At 5 dpi, broiler chickens were greatly reduced in beneficial bacteria such as Lactobacillus, and the number of opportunistic pathogens increased, especially the conditional pathogens Escherichia abundance exceeded 40%. Similarly, H9N2 AIV infected mice also showed some changes in bacterial population, resulting in increased abundance of Proteobacteria and Actinomycetes. Especially, beneficial Lactobacillus was significantly down-regulated, and Streptococcus, Staphylococcus and Corynebacterium-1 were significantly up-regulated at 12 dpi. As we know, the etiological relationship between gastrointestinal diseases and intestinal microflora was relatively constant [22, 23]. We proposed that the structure disruption of mouse intestinal flora caused by H9N2 AIV probably took shape a bacterial environment to trend to critical pathological conditions. Meanwhile, we isolated plenty of Staphylococcus and a few E. coli from liver and lung in infected mice. Those findings provide clues that these microbes might be from translocation of intestine. The isolation of Neongreen-labeled bacteria confirmed this hypothesis. The labeled bacteria transferred from the intestine through the intestinal epithelial tissue to the mesenteric tissue sites in 36 h and lungs in 24 h post intragastrical administration of Neongreen-labeled bacteria. These results indicated that H9N2 AIV promotes some bacterium translocation into body, then invaded other tissues through body fluid circulation and so on, leading to bacterial infection. Which bacteria can synergize with H9N2 AIV co-infection and how does this synergy work? These require further study. In addition, the labeled bacteria were also isolated from lungs of the infected mouse group 24 h post intragastrical administration, indicating specific intestinal bacteria may also be able to infect damaged lungs by the digestive tract.

Patchouli is an edible plant, which broadly grows in China and is the main ingredient of AL. As one of the 50 fundamental herbs, Patchouli is used in popular medicine to viral, fungal, and bacterial infections [24‐26]. Magnolia officinalis (Chinese name: Houpo) was an important constituent of AL and rich in Honokiol or magnolol, isomers of neolignans [27, 28], which was reported that honokiol have function of anti-oxidative, anti-inflammatory, anti-tumor and anti-microbial properties [29‐34]. A previous review showed that the mechanisms of kampo medicines including some components of AL were involved in regulating the intestinal motility by NO or 5-HT3 receptor pathways and anti-inflammatory [35]. As a thousand-year-old formula, AL combines each component biological and pharmacological property and is an efficiency and low toxicity natural medicines. It is reported that the AL can increase the number of Lactobacillus, Bifidobacterial in mammal gut, reduce intestinal permeability in acetic acid-induced PI-IBS, regulate CD4+ and CD8+ cells in Peyer’s patch and suppress TNF-alpha levels in enteric homogenates to improve the diarrhea caused by Salmonella typhimurium in mice [9, 10, 36, 37, 38]. Our experiment found AL can effectively minimize the H9N2 AIV infection-induced intestinal mucosal injuries and alleviate bacterial flora disorder, suggesting that AL no only might somehow improve the immunity and anti-H9N2 AIV capabilities of host, but also repair intestinal barrier to prevent secondary infections. Thus, as a low resistance and toxicity natural medicine, traditional Chinese medicine has many pharmacological activities and could be applied to treat many diseases as potential antibiotic substitutes. Those need to further research.

Meanwhile, little to no bacteria was isolated from liver and lung of mice after H9N2 AIV infection in Ageratum-liquid group and Infection-Ageratum-liquid group. Moreover, no Neongreen-labeled bacteria were isolated from liver, lung, and mesentery tissues of the mice from both of the treatment groups, but the Neongreen-labeled bacteria were isolated from the intestines of all mice subjected to either administration of Neongreen-labeled bacteria or H9N2 AIV infection followed by administration of Neongreen-labeled bacteria, indicating that gut bacterial translocation to the baby might play important roles during H9N2 AIV infection process and AL treatment effectively prevented the bacteria translocation that otherwise would be induced post H9N2 AIV infection in mice.

As a low-virulent virus, AIV H9N2 causes mild respiratory illness, but usually involved secondary bacterial infection in mammals and chicken [1, 3, 5]. Our study explicitly demonstrated that H9N2 AIV infection could damage the ileal mucosal barrier and induce the disturbance of the intestinal flora in BALB/c mice resulting in translocation of intestinal bacteria. Our data also illustrated that the traditional Chinese medicine AL can effectively prevent bacterial translocation following H9N2 infection. These findings are of important theoretical and practical significance in minimizing H9N2 AIV clinical damages, including secondary infections.