Maternal imprinting of the neonatal microbiota colonization in intrauterine growth restricted piglets:a review

Early colonization of intestinal microbiota during the neonatal stage plays an important role on the development of intestinal immune system and nutrients absorption of the host. Compared to the normal birth weight(NBW)piglets, intrauterine growth restricted(IUGR) piglets have a different intestinal microbiota during their early life,which is related to maternal imprinting on intestinal microbial succession during gestation, at birth and via suckling.Imbalanced allocation of limited nutrients among fetuses during gestation could be one of the main causes for impaired intestinal development and microbiota colonization in neonatal IUGR piglets. In this review, we summarized the potential impact of maternal imprinting on the colonization of the intestinal microbiota in IUGR piglets, including maternal undernutrition, imbalanced allocation of nutrients among fetuses, as well as vertical microbial transmission from mother to offspring during gestation and lactation. At the same time, we give information about the current maternal nutritional strategies(mainly breastfeeding, probiotics and prebiotics) to help colonization of the advantageous intestinal microbiota for IUGR piglets.     

猪肠道微生物及其代谢产物与肠道屏障研究进展

肠道微生物参与营养物质代谢，影响猪的健康和发育，当肠道微生物发生紊乱，会造成猪腹泻并引起炎症反应，因此肠道微生物对猪的健康起着至关重要的作用。本文从肠道微生物在仔猪不同发育阶段的分布、肠道微生物的代谢产物对肠道健康的影响机制和肠道微生物与肠道屏障之间的关系进行阐述，并探讨了目前肠道健康研究的进展以及今后的研究方向，旨在为猪肠道健康调控提供理论参考。     

Concurrent porcine rotaviral and transmissible gastroenteritis viral infections in a three-day-old conventional pig.

A 3-day-old suckling pig with diarrhea was necropsied, and immunofluorescent microscopic examination of the small intestinal mucosa, together with immune electron microscopic examination of the large intestinal contents, provided a presumptive diagnosis of a concurrent infection with transmissible gastroenteritis (TGE) virus and porcine rotavirus. Immunofluorescent microscopic, immune electron microscopic, and serologic data obtained from gnotobiotic pigs experimentally inoculated with the large intestinal contents of the suckling pig confirmed this diagnosis. Two gnotobiotic pigs, convalescent from previous TGE viral infections, became infected with porcine rotavirus only. However, another gnotobiotic pig, convalescent from a previous porcine rotaviral infection, became infected with TGE virus only, following inoculation with the large intestinal contents of the suckling pig.     

Intestinal microbiota differentially affect brush border enzyme activity and gene expression in the neonatal gnotobiotic pig

To study microbial influence on intestinal development pertaining to nutrient digestion, two separate gnotobiotic experiments were performed, each with 16 piglets allocated to four treatment groups: germfree (GF), monoassociation with Escherichia coli, monoassociation with Lactobacillus fermentum or conventionalization with faecal bacteria (CV). Enzyme activity and gene expression of lactase phlorizin hydrolase (LPH) and aminopeptidase N (APN) were measured in isolated enterocytes, harvested on day 14, using specific substrates and quantitative PCR respectively. Enterocytes of CV pigs had reduced APN activity, but had increased gene expression relative to GF, making the specific activity:mRNA (A:G) ratio dramatically lower (p < 0.05). Similarly, LPH A:G ratio was significantly reduced (p < 0.05) in enterocytes of CV pigs as compared with GF. The results of co‐incubation of L. fermentum, E. coli and faecal bacteria with APN indicate a direct relationship between enzyme inactivation and specific A:G ratio in enterocytes. We conclude that enterocyte up‐regulation of APN expression occurs as either a direct response to microbial colonization or as a feedback mechanism in response to reduced enzyme activity through microbial degradation. This mechanism may play a role in ensuring effective competition of the host with the intestinal microbiota for available nutrients.     

Lectin binding profile of the small intestine of five-week-old pigs in response to the use of chlortetracycline as a growth promotant and under gnotobiotic conditions

Antibiotics have traditionally been used for growth promotion in the pork industry; however, their use in animal feed has recently been limited because of human health concerns. The intestinal microbiota plays an important role in mediating many physiological functions such as digestion and animal growth. It was hypothesized that use of antibiotics as growth promotants and subsequent variations in intestinal microbiota induce significant changes in the intestinal glycoconjugate composition, which ultimately affects animal growth and disease susceptibility. The aim of this study was to characterize the lectin binding profiles of the ileum of weanling pigs in response to the absence of intestinal microbiota and to the use of the antibiotic chlortetracycline as growth promotant. Eighteen half-sib piglets obtained by cesarean section were divided into 3 treatment groups (n = 6) and maintained as control, antibiotic-fed, and gnotobiotic piglets until 5 wk of age. The glycoconjugate composition of the ileal tissues was examined by lectin histochemistry. Lycopersicon esculentum lectin, Jacalin, Pisum sativum agglutinin, Lens culinaris agglutinin (LCA), and Sambucus nigra lectin showed significant differences (P < 0.05) in binding intensities on the dome and villous epithelium between the treatment groups. Griffonia simplicifolia lectin I, Glycine maxi agglutinin, and Arachis hypogea agglutinin exhibited differences (P < 0.05) between treatment groups in lectin binding on goblet cells. Triticum vulgaris agglutinin, Pisum sativum agglutinin, and LCA showed significant differences (P < 0.05) in binding intensities on dome, corona, and follicular regions of the ileum among treatment groups of animals. Overall, ileal tissues from gnotobiotic piglets expressed significantly weaker (P < 0.05) lectin binding for many lectins compared with control and antibiotic groups. This suggests that the intestinal microbiota plays an important role in the expression of sugar moieties in the intestine. Lectins LCA, Phaseolus vulgaris Leucoagglutinin, and Maackia amurensis lectin II showed significant differences (P < 0.05) in lectin bindings between control and antibiotic-fed piglets. This indicates that chlortetracycline as a growth promotant induces biologically relevant changes in the lectin binding profile of the ileum. These findings will help in further understanding the role of the gut microbiota and the mechanisms of action of antibiotics as growth promotants in pigs.     

仔猪早期肠道微生物变化特点与饲养管理

仔猪早起肠道微生物菌群的变化对于仔猪的生长发育有重要的作用。为提高仔猪的存活率,在了解仔猪早期肠道微生物变化特点的基础上,采取科学的饲养管理措施,为仔猪提供干净、舒适的生长环境,保障仔猪的健康生长发育具有重要的科学价值和应用价值。该文主要论述仔猪早期肠道微生物变化特点出发,提出一套仔猪的饲养管理方式,为仔猪的饲养管理提供可靠的技术支持。     

断奶仔猪肠道健康与免疫调控

断奶应激一直是困扰现代养猪业的难题,由于幼龄仔猪消化系统和免疫系统都尚未发育成熟,早期断奶容易造成仔猪胃肠道功能紊乱,诱发仔猪断奶综合征。肠道是断奶仔猪消化食物、吸收营养的主要场所,也是体内最大的免疫器官之一。因此,通过研究仔猪肠道健康,阐明仔猪肠道生理结构及功能的发育规律,分析仔猪断奶前后肠道免疫调控特点,为不同日龄断奶仔猪确定合理的营养对策具有十分重要的意义,文章就近年来国内外仔猪肠道生理及免疫调控的研究进展进行综述。     

Influence of probiotic Lactobacilli colonization on neonatal B cell responses in a gnotobiotic pig model of human rotavirus infection and disease

The goal of this study was to define the impact of colonization of gnotobiotic (Gn) pigs with lactic acid bacteria (LAB) on development of intestinal and systemic B cell responses to human rotavirus (HRV). The LAB-specific and total B cell responses were also assessed. Gn pigs were inoculated with LAB (Lactobacillus acidophilus and L. reuteri) and virulent Wa strain HRV (LAB+HRV+), HRV only (LAB-HRV+), LAB only (LAB+HRV-) or mock (LAB-HRV-). The HRV infection induced similar HRV-specific intestinal and systemic antibody and B cell responses in pigs with or without LAB, whereas LAB significantly enhanced total intestinal IgA secreting cell responses and total serum IgM and intestinal IgM and IgG titers. The LAB colonization did not reduce HRV shedding or diarrhea, this may be partly due to the short time interval between the first LAB feeding and HRV inoculation. Further studies are needed with longer time for LAB to establish before HRV inoculation. However, our studies demonstrate that Gn pigs infected with HRV develop a similar magnitude of virus-specific B cell responses as those of HRV-infected and LAB colonized pigs. LAB colonization alone is not as efficient in promoting intestinal B cell responses, as is HRV infection.     

Lactic acid bacterial colonization and human rotavirus infection influence distribution and frequencies of monocytes/macrophages and dendritic cells in neonatal gnotobiotic pigs

Despite accumulating knowledge of porcine macrophages and dendritic cells (DCs) from in vitro studies, information regarding monocytes/macrophages and DCs in lymphoid tissues of enteric pathogen-infected neonatal animals in vivo is limited. In this study we evaluated the influence of commensal bacterial [two strains of lactic acid bacteria (LAB), Lactobacillus acidophilus and L. reuteri] colonization and rotavirus infection on distribution and frequencies of monocytes/macrophages and conventional DCs (cDCs) in ileum, spleen and blood. Gnotobiotic pigs were inoculated with LAB and virulent Wa strain human rotavirus (HRV) (LAB+HRV+), HRV only (LAB-HRV+), LAB only (LAB+HRV-) or mock (LAB-HRV-). The cDCs were characterized as SWC3(+)CD11R1(+), whereas monocytes/macrophages were identified as SWC3(+)CD11R1(-) by flow cytometry in the gnotobiotic pigs at 10 days of age. Infection with HRV alone activated/recruited significantly more monocytes/macrophages to the intestine than LAB colonization and 56% versus 28% of these cells expressed CD14. Colonization with LAB alone also significantly increased the frequencies of monocytes/macrophages and cDCs and the CD14 expression on monocytes/macrophages in ileum and spleen compared to the controls. LAB colonization plus HRV infection significantly reduced macrophage and cDC frequencies in spleen compared to LAB colonization or HRV infection alone, suggesting that LAB colonization down-regulated HRV- infection-induced monocyte/macrophage activation/recruitment at the systemic lymphoid tissue. These results illustrated the distribution of porcine monocytes/macrophages and cDCs and the frequencies of CD14 expression on these cells in intestinal and systemic lymphoid tissues in the early stage of immune responses to intestinal colonization by LAB versus infection by an enteric pathogen HRV and will facilitate further in vivo studies on functional characterization of these immune cells in neonates.     

The influence of omega-3 polyunsaturated fatty acids (omega-3 pufa) on lactobacilli adhesion to the intestinal mucosa and on immunity in gnotobiotic piglets

The effect of application of omega-3 polyunsaturated fatty acids (omega-3 PUFA) on intestinal colonization by Lactobacillus paracasei and on cellular immunity has been investigated in gnotobiotic pigs. The administration of polyunsaturated fatty acids positively affected the adhesion of Lactobacillus paracasei to the jejunal mucosa of gnotobiotic piglets. When compared to the control group, the number of Lactobacillus paracasei adhering to the jejunal mucosa was by 12% higher in piglets of the experimental group (5.10 log 10/cm2 vs. 4.55 log 10/cm2). The respective counts of Lactobacillus paracasei adhering to the ileal and colonic mucosa of 28 day old gnotobiotic piglets reached 4.45 and 5.05 log 10/cm2 in group C and 4.44 and 4.95 log 10/cm2 in group E. Omega-3 PUFA supplementation increased the phagocytic activity of neutrophils by almost 100% on day 28 of life as well as the subpopulations of lymphocytes (CD8) in the peripheral blood of germ-free piglets on day 21 of life. Our results indicate that the action of probiotics in the gut may be modulated by dietary PUFA. The stimulatory effect of PUFA upon adhesion of lactobacilli could be used for enhancing the effectiveness of probiotics in inhibiting digestive tract pathogens.     

Piglet gut microbial shifts early in life:causes and effects

The gut microbiome has long been known to play fundamentally important roles in the animal health and the well-being of its host. As such, the establishment and maintenance of a beneficial gut microbiota early in life is crucial in pigs, since early gut colonizers are pivotal in the establishment of permanent microbial community structures affecting the health and growth performance of pigs later in life. Emphasizing this importance of early gut colonizers, it is critical to understand the factors impacting the establishment of the piglet gut microbiome at weaning. Factors include, among others, diet, in-feed antibiotics, probiotics and prebiotic administration. The impact of these factors on establishment of the gut microbiome of piglets at weaning includes effects on piglet gut microbial diversity, structure, and succession. In this review, we thoroughly reviewed the most recent findings on the piglet gut microbiome shifts as influenced by weaning, and how these microbiome changes brought about by various factors that have been shown to affect the development of microbiota in piglets. This review will provide a general overview of recent studies that can help to facilitate the design of new strategies to modulate the gut microbiome in order to enhance gastrointestinal health, growth performance and well-being of piglets.     

Insights into host-microbe interaction:What can we do for the swine industry?

Recent discoveries have underscored the cross-talk between intestinal microbes and their hosts. Notably, intestinal microbiota impacts the development, physiological function and social behavior of hosts. This influence usually revolves around the microbiota-gut-brain axis (MGBA). In this review, we firstly outline the impacts of the host on colonization of intestinal microorganisms, and then highlight the influence of intestinal microbiota on hosts focusing on short-chain fatty acid (SCFA) and tryptophan metabolite-mediated MGBA. We also discuss the intervention of intestinal microbial metabolism by dietary supplements, which may provide new strategies for improving the welfare and production of pigs. Overall, we summarize a state-of-the-art theory that gut microbiome affects brain functions via metabolites from dietary macronutrients.     

Intestinal microbiota and its interaction to intestinal health in nursery pigs

The intestinal microbiota has gained increased attention from researchers within the swine industry due to its role in promoting intestinal maturation,immune system modulation,and consequently the enhancement of the health and growth performance of the host.This review aimed to provide updated scientific information on the interaction among intestinal microbiota,dietary components,and intestinal health of pigs.The small intestine is a key site to evaluate the interaction of the microbiota,diet,and host because it is the main site for digestion and absorption of nutrients and plays an important role within the immune system.The diet and its associated components such as feed additives are the main factors affecting the microbial composition and is central in stimulating a beneficial population of microbiota.The microbiotaehost interaction modulates the immune system,and,concurrently,the immune system helps to modulate the microbiota composition.The direct interaction between the microbiota and the host is an indication that the mucosa-associated microbiota can be more effective in evaluating its effect on health parameters.It was demonstrated that the mucosa-associated microbiota should be evaluated when analyzing the interaction among diets,microbiota,and health.In addition,supplementation of feed additives aimed to promote the intestinal health of pigs should consider their roles in the modulation of mucosa-associated microbiota as biomarkers to predict the response of growth performance to dietary interventions.     

Immunity to Escherichia coli in Pigs. The Role of Milk in Protective Immunity to E. coli Enteritis

Milk whey from immunized sows increased the survival time of gnotobiotic piglets infected with Escherichia coli. The survival time of infected piglets fed milk whey from non-vaccinated sow's was the same as that of similar pigs fed condensed cow milk.

The significance of milk antibodies in immune protection against E. coli enteritis is discussed and compared with that of absorbed colostral antibodies. From calculations presented it would appear that seven day old piglets receive approximately 1 g of gamma globulin daily from milk and that this is equal to the piglets total serum gamma globulin content. After seven days of age the gamma globulin content of piglet serum falls, whereas that of milk remains constant; milk is, therefore, potentially a major source of immunoglobulins with protective activity against E. coli associated enteritides.

     

Experimental enteric infection of gnotobiotic piglets with a Chlamydia psittaci strain of avian origin

The pathogenicity of a Chlamydia psittaci isolate of pigeon origin was assessed using a litter of gnotobiotic piglets. At 3 days of age, six piglets were inoculated intragastrically with egg-grown chlamydiae, the remaining six pigs were sham-inoculated. The animals were observed for clinical signs, and they were killed and necropsied sequentially between 4 and 15 days of age. Clinical manifestations consisted of slight softening of the faeces between 6 and 10 days post-inoculation (DPI). Immunohistochemistry revealed chlamydial replication predominantly in the small intestine, initially within villous enterocytes, after 4 DPI mostly in the lamina propria. Histopathology showed villous atrophy and increased numbers of inflammatory cells in the gut up to 6 DPI. Chlamydial stages of normal morphology were identified within enterocytes using transmission electron microscopy. An enzyme-linked immunosorbent assay (ELISA) run on faecal samples revealed shedding of chlamydial antigen from 3 until 11 DPI. Systemic dissemination of Chlamydia occurred to a limited extent according to polymerase chain reaction and immunohistochemistry results of several extraintestinal organs. Corresponding histopathological changes were minimal. Sera of all pigs were negative for anti-chlamydial antibodies using a complement fixation test. In conclusion, inoculation of this isolate in gnotobiotic piglets resulted in a productive enteric infection with mild lesions, weak systemic dissemination, and faecal shedding, indicating the pig as a potential host for avian chlamydiae.     

ESCHERICHIA COLI AND ROTAVIRUS INFECTIONS IN FOUR-WEEKOLD GNOTOBIOTIC PIGLETS FED MILK OR DRY FOOD

A haemolytic enteropathogenic E. coli (WG) and pig rotavirus were isolated from a field case of postweaning diarrhoea in pigs. Four-week-old gnotobiotic piglets fed on milk diet were found to be extremely susceptible to infection with WG E. coli . Piglets were less susceptible to the infection immediately after the diet was changed from milk to dry food, and were almost completely resistant 4 days after the change to dry food. There was no difference in the clinical response to infection with WG E. coli when the piglets were fed either a high energy diet or low energy diet. Four-week-old piglets fed milk showed mild symptoms of diarrhoea when inoculated with pig rotavirus. Symptoms were more severe when piglets were inoculated immediately after the change from milk to dry food. Piglets inoculated 4 days after the change of diet showed no symptoms of diarrhoea at all. Under the conditions of these experiments the enteropathogenic E. col produced a more serious disease than did pig rotavirus. Infection of 4-week-old gnotobiotic piglets with both agents given sequentially produced a diarrhoeal disease that was more severe than that produced by each agent separately.     

Experimental colonization of piglets and gilts with systemic strains of Haemophilus parasuis and Streptococcus suis to prevent disease.

Haemophilus parasuis and Streptococcus suis are both major causes of losses during the nursery period, especially in herds using the segregated early weaning system. In this system, only a few piglets may be colonized with the herd's prevalent systemic strain, which results in infection of naive penmates late in the nursery. In view of these factors, the objectives of this study were: (1) to evaluate the early colonization of piglets with the farm's prevalent systemic strain of H. parasuis and S. suis as an alternative method for disease prevention; and (2) to evaluate 2 different protocols for experimental colonization: direct colonization of piglets and colonization of piglets through nose-to-nose contact with inoculated sows. Haemophilus parasuis and S. suis isolates recovered from diseased nursery pigs were characterized by the rep-PCR technique and the herd's prevalent strains were used for colonization. Piglets in the experimentally colonized groups were inoculated at 5 days of age by the oral route using a spray pump. Sows were colonized at 2 weeks prior to farrowing using a similar protocol. Although both colonization protocols were successful in getting the piglets colonized, direct inoculation of 5-day-old piglets with the herd's systemic strains of H. parasuis and S. suis tended to be more effective in reducing the morbidity and the mortality than the colonization of piglets by nose-to-nose contact with inoculated sows.     

Emigration of polymorphonuclear leucocytes into the intestinal lumen of the neonatal piglet in response to challenge with K88-positive Escherichia coli

The emigration of neutrophils from the blood of neonatal piglets into the intestinal lumen in response to a K88-positive strain of Escherichia coli was investigated. The pig herd used was of known genetic susceptibility to K88-positive E coli and had recently experienced an outbreak of neonatal diarrhoea. Neutrophil emigration depended on certain factors. Neutrophils emigrated into ligated loops in susceptible piglets sucking immune colostrum from susceptible dams but not into loops in colostrum deprived resistant piglets or piglets sucking non-immune colostrum from resistant dams. In susceptible, colostrum deprived piglets neutrophils in intestinal contents were only associated with severe lesions. Large numbers of neutrophils which appeared at several foci on the villi were observed in three of six resistant piglets that sucked immune colostrum from susceptible dams. It was concluded that neutrophil emigration into the intestinal lumen of piglets could occur in response to K88-positive E coli and resulted not from the presence or absence of the intestinal K88-receptor but from the ingestion of immune colostrum.     

Pathogenesis of porcine enteric calicivirus-like virus in four-day-old gnotobiotic pigs

Eighteen 4-day-old gnotobiotic pigs were orally inoculated with porcine enteric calicivirus-like virus (C strain). Seven additional gnotobiotic pigs served as noninoculated controls. Mild diarrhea developed in all inoculated pigs by postinoculation day (PID) 3 and persisted for 3 to 7 days. Severe diarrhea developed in 2 inoculated pigs between PID 4 and 5. Twelve inoculated and 7 control pigs were euthanatized over a 7-day period. Small intestinal mucosal smears were stained with a fluorescein-conjugated anti-porcine enteric calicivirus-like virus serum. Immunofluorescence was observed in villous epithelial cells (primarily in the duodenum or jejunum) of all inoculated pigs, except for 1 pig euthanatized at PID 7. Villus length was determined in histologic sections of the small intestinal specimens from control and inoculated pigs. Statistically significant (P less than 0.01) villus atrophy was found in the duodenum and/or jejunum of inoculated pigs at PID 3 to 7. These observations were confirmed by scanning electron microscopy, which revealed shortening, blunting, fusion, or absence of villi in the duodenum and jejunum of inoculated pigs at PID 3 to 7. Lesions were not seen in control pigs. Calicivirus-like particles were detected by immune electron microscopy in the large intestinal contents and feces of inoculated pigs from PID 1 to 7.     

Dietary fibre enrichment of supplemental feed modulates the development of the intestinal tract in suckling piglets

Background: Commercial pre-weaning diets are formulated to be highly digestible and nutrient-dense and contain low levels of dietary fibre. In contrast, pigs in a natural setting are manipulating fibre-rich plant material from a young age. Moreover, dietary fibre affects gastrointestinal tract(GIT) development and health in older pigs. We hypothesised that supplemental diets that contain vegetal fibres are accelerating GIT development in suckling piglets in terms of size and functionality. From d 2 of life, sow-suckled piglets had access to a low fibre diet(CON),a diet with a fermentable long-chain arabinoxylan(lc-AXOS), a diet with a largely non-fermentable purified cellulose(CELL), or a diet containing both fibres. During the initial 2 weeks, the control diet was a high-density milk replacer,followed by a dry and highly digestible creep meal. Upon weaning at 25 d, 15 piglets from each treatment group,identified as eaters and originating from six or seven litters, were sacrificed for post-mortem examination of GIT morphology, small intestinal permeability and metabolic profile of the digesta. The microbiota composition of the mid-colon was evaluated in a sub-set of ten piglets.Results: No major statistical interactions between the fibre sources were observed. Piglets consumed the fibrecontaining milk supplements and creep diets well. Stomach size and small intestinal permeability was not affected.Large intestinal fill was increased with lc-AXOS only, while relative large intestinal weight was increased with both fibre sources(P 0.050). Also, CELL decreased ileal pH and tended to increase ileal DM content compared to CON(P 0.050). Moreover, the concentration of volatile fatty acids was increased in the caecum(P 0.100) and midcolon(P 0.050) by addition of CELL. lc-AXOS only stimulated caecal propionate(P 0.050). The microbiota composition showed a high individual variation and limited dietary impact. Nonetheless, CELL induced minor shifts in specific genera, with notable reductions of Escherichia-Shigella.Conclusions: Adding dietary fibres to the supplemental diet of suckling piglets altered large intestinal morphology but not small intestinal permeability. Moreover, dietary fibre showed effects on fermentation and modest changes of microbial populations in the hindgut, with more prominent effects from the low-fermentable cellulose.