Establishment in the piglet gut of lactobacilli capable of degrading mixed-linked β-glucans

The establishment in piglets of lactobacilli with ability to degrade mixed-linked (1 → 3), (1 → 4) β-D-glucans was studied in faeces from 15 animals. The piglets had free access to creep feed with an estimated content of 2%β-d-glucans from 5 days of age. On days 3 and 35, ca log 8 cfu/g of β-glucan-degrading lactobacilli were found in a majority of the samples. On days 7, 14 and 21 such bacteria were only found occasionally.
During establishment of the microflora in the neonate, the faecal lactobacilli of the piglet seemed related to those of the sow. Later, the metabolic activity of the lactobacilli in piglet faeces showed a connection to the composition of the diet. The possible relation of these bacteria to occurrence of β-glucanases attributed to be endogenous in the pig is discussed.     

Development of intestinal lactobacilli in normal piglets

The development of intestinal Lactobacillus spp. in 18 normal piglets from six litters was investigated from 1 to 45 d after birth. Faecal Lactobacillus strains isolated from piglets and their dams were identified at species and biovar levels on the basis of carbohydrate fermentation. A definite pattern was observed in the changes of four species and 20 biovars of lactobacilli appearing in faeces, with growth of the piglets. Lactobacillus reuteri colonized the animals on the first day of birth. The Lact. acidophilus group appeared 1 week after birth and maintained high levels in the intestine. Biovars B, C and E of Lact. reuteri were recovered as major biovars on the first to fourteenth days after birth. In the third and fourth weeks after birth, however, biovars F and G of Lact. reuteri were mainly recovered from piglet faeces. By the seventh week, biovars B, C and E predominated again in the intestine. No change in the composition of species and biovars of the genus Lactobacillus in the dams was detected.     

Characterization of dominant cultivable lactobacilli and their antibiotic resistance profiles from faecal samples of weaning piglets

AIMS: To examine the lactic acid bacteria flora of weaning piglets, to define the distribution of different lactobacilli species in piglet faecal samples, and to determine the susceptibility phenotype to 11 antibiotic of different families. METHODS AND RESULTS: The faecal samples were taken from piglets with good herd status at 11 and 28 days after weaning. The Lactobacillus isolates (n = 129) from 78 animals housed in pairs in 39 pens were preliminarily identified by their morphology and biochemical characteristics. Partial 16S ribosomal DNA (16S rDNA) was used to identify the isolates to the species level, and RAPD (randomly amplified polymorphism DNA) profiles to differentiate Lactobacillus isolates to the strain level. Based on these studies, 67 strains were selected for antibiotic resistant tests. The most numerous Lactobacillus species found in the piglets was Lactobacillus reuteri (n = 43). Other lactobacilli were L. salivarius (n = 15), L. agilis (n = 4), L. johnsonii (n = 2), L. vaginalis (n = 1), L. mucosae (n = 1) and L. gallinarum (n = 1). All the strains were susceptible to chloramphenicol, ampicillin and gentamicin. Two L. salivarius isolates and two L. reuteri isolates were found to be multiresistant. CONCLUSIONS: This study indicates that the faecal Lactobacillus flora in piglets consists mainly of L. reuteri, L. salivarius and L. acidophilus group lactobacilli, and the distribution of lactobacilli is similar between individuals of the same age and with the same diet. Most of the Lactobacillus isolates tested were sensitive to the antibiotics used in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Valuable information on Lactobacillus species distribution and their antibiotic resistance profiles in piglets is obtained.     

益生菌Lactobacillus amylovorus S1对仔猪后肠菌群的影响

结合PCR/DGGE(Denaturinggradientgelelectrophoresis,变性梯度凝胶电泳)和16SrDNA序列分析技术,研究添加益生菌LactobacillusamylovorusS1后仔猪从7至35日龄(断奶后两周)后肠菌群的变化。6窝新生仔猪被随机分成两组:对照组和处理组,处理组仔猪于7、9和11日龄口服L.amylovorusS1菌液(活菌数5×109CFU/mL)。分别于7、14、21、24和35日龄,每窝随机屠宰一头仔猪,收集肠道样品。比较不同日龄仔猪后肠菌群DGGE图谱表明,断奶后图谱中多数高GC含量细菌条带消失,至断奶后两周又逐渐出现。序列分析显示,这些高GC含量细菌主要为乳酸杆菌。统计分析表明,仔猪口服益生菌S1对其盲肠和结肠菌群的多样性指数无显著影响。通过比较处理组和对照组图谱发现,处理组14日龄出现一特异条带,与其匹配的序列的最相似已知菌为Clostridiumdisporicum,相似性为95%;而35日龄对照组有一特异优势条带,该条带被鉴定为猪链球菌(Streptococcussuis),相似性为99%。     

16S ribosomal RNA-based methods to monitor changes in the hindgut bacterial community of piglets after oral administration of Lactobacillus sobrius S1

16S ribosomal RNA (rRNA) gene based PCR/denaturing gradient gel electrophoresis (DGGE) and real-time PCR were used to monitor the changes in the composition of microbiota in the hindgut of piglets after oral administration of Lactobacillus sobrius S1. Six litters of neonatal piglets were divided randomly into control group and treatment group. At 7, 9, and 11 days of age, piglets in the treatment group orally received a preparation of L. sobrius S1. At 7, 14, 21(weaning), 24, and 35 days of age, one piglet from each litter was sacrificed and digesta samples of hindgut were collected. DGGE analysis of 16S rRNA gene V6-V8 region for all bacteria showed that several populations present in the hindgut of piglets, represented by far-migrating bands, disappeared after weaning. Most of these bands corresponded to Lactobacillus spp. as revealed by sequence analysis. Quantitative real-time PCR specific for lactobacilli further demonstrated that the number of lactobacilli population tended to decrease after the piglets were weaned. Drastic changes of L. amylovorus and L. sobrius in total Lactobacillus populations were also observed in the colon of piglets around weaning, as monitored by 16S rRNA gene V2-V3 region based Lactobacillus-specific PCR-DGGE. Species-specific real-time PCR also revealed that the population of L. sobrius declined apparently in the colon of piglets after weaning. No remarkable changes in the overall microbial community in the hindgut were found between control and treatment groups. However, comparison of DGGE profiles between the two groups revealed a specific band related to Clostridium disporicum that was found in treatment group on day 14. On day 35, a specific band appeared only in the control group, representing a population most closely related to Streptococcus suis (99%). Real-time PCR showed that L. sobrius 16S rRNA gene copies in treatment group were relatively higher than in the control group (10(8.45) vs. 10(6.83)) on day 35, but no significant difference was observed between the two groups.     

Colonization of the porcine gastrointestinal tract by lactobacilli

Eight strains of lactobacillus isolated from the porcine gastrointestinal tract were tested for their ability to adhere in vitro to cells collected from stratified squamous epithelium in the digestive tracts of newborn piglets. Piglets were inoculated with individual strains, and their digestive tracts were sampled at intervals to determine the colonizing ability of the lactobacilli. The results of the in vitro test did not predict whether a lactobacillus strain would associate with stratified squamous epithelium in the piglet digestive tract, but epithelial association in vivo appeared to be an important factor in the maintenance of lactobacillus populations in the tract. None of the lactobacillus strains used as inocula was numerically dominant in the tract 7 days after inoculation of the piglets with a single dose of the bacteria.     

Colonization of the porcine gastrointestinal tract by lactobacilli.

Eight strains of lactobacillus isolated from the porcine gastrointestinal tract were tested for their ability to adhere in vitro to cells collected from stratified squamous epithelium in the digestive tracts of newborn piglets. Piglets were inoculated with individual strains, and their digestive tracts were sampled at intervals to determine the colonizing ability of the lactobacilli. The results of the in vitro test did not predict whether a lactobacillus strain would associate with stratified squamous epithelium in the piglet digestive tract, but epithelial association in vivo appeared to be an important factor in the maintenance of lactobacillus populations in the tract. None of the lactobacillus strains used as inocula was numerically dominant in the tract 7 days after inoculation of the piglets with a single dose of the bacteria.     

Roles of Probiotic Lactobacilli Inclusion in Helping Piglets Establish Healthy Intestinal Inter-environment for Pathogen Defense

The gastrointestinal tract of pigs is densely populated with microorganisms that closely interact with the host and with ingested feed. Gut microbiota benefits the host by providing nutrients from dietary substrates and modulating the development and function of the digestive and immune systems. An optimized gastrointestinal microbiome is crucial for pigs’ health, and establishment of the microbiome in piglets is especially important for growth and disease resistance. However, the microbiome in the gastrointestinal tract of piglets is immature and easily influenced by the environment. Supplementing the microbiome of piglets with probiotic bacteria such as Lactobacillus could help create an optimized microbiome by improving the abundance and number of lactobacilli and other indigenous probiotic bacteria. Dominant indigenous probiotic bacteria could improve piglets’ growth and immunity through certain cascade signal transduction pathways. The piglet body provides a permissive habitat and nutrients for bacterial colonization and growth. In return, probiotic bacteria produce prebiotics such as short-chain fatty acids and bacteriocins that benefit piglets by enhancing their growth and reducing their risk of enteric infection by pathogens. A comprehensive understanding of the interactions between piglets and members of their gut microbiota will help develop new dietary interventions that can enhance piglets’ growth, protect piglets from enteric diseases caused by pathogenic bacteria, and maximize host feed utilization.     

变性梯度凝胶电泳法研究断奶仔猪粪样细菌区系变化

利用PCR和DGGE技术分析了12头仔猪在断奶后其粪样细菌区系的变化。粪样细菌16S rDNA的V6～V8可变区经PCR扩增,扩增产物经DGGE电泳后再进行相似性分析。结果表明,仔猪断奶当天粪样 DGGE谱带少,同窝仔猪间图谱相似。断奶后,随着断奶时间的推移,每头仔猪的DGGE图谱带逐渐增多,变得复杂和多样,仔猪个体间DGGE图谱差异逐渐增大。仔猪是否同窝以及所采食日粮类型对DGGE图谱没有明显影响。相似性分析还表明,日粮中添加寡果糖的仔猪在断奶后第1周,其粪样微生物区系变化迅速,而后缓慢。     

Lactobacillus succession in the piglet digestive tract demonstrated by plasmid profiling

Plasmid profiling was used to distinguish strains of lactobacilli inhabiting the digestive tract of piglets and the feces of sows. Fifteen plasmid profile types were detected among 328 isolates of lactobacilli. Plasmid profiling of lactobacilli permitted the following conclusions to be made: the maternal feces were a major source of lactobacilli colonizing the piglet digestive tract; the lactobacillus population of the gastric region of the piglet digestive tract was composed of lactobacillus strains different from those present in the rectal population; and a lactobacillus succession was observed in the digestive tract of piglets drawn from a single litter, and one plasmid profile type became dominant in the gastric region of these animals.     

Lactobacillus succession in the piglet digestive tract demonstrated by plasmid profiling.

Plasmid profiling was used to distinguish strains of lactobacilli inhabiting the digestive tract of piglets and the feces of sows. Fifteen plasmid profile types were detected among 328 isolates of lactobacilli. Plasmid profiling of lactobacilli permitted the following conclusions to be made: the maternal feces were a major source of lactobacilli colonizing the piglet digestive tract; the lactobacillus population of the gastric region of the piglet digestive tract was composed of lactobacillus strains different from those present in the rectal population; and a lactobacillus succession was observed in the digestive tract of piglets drawn from a single litter, and one plasmid profile type became dominant in the gastric region of these animals.     

Piglet growth before and after weaning in relation to a qualitative estimate of solid (creep) feed intake during lactation: a pilot study

The experimental objectives were to verify whether a qualitative measure of creep feed consumption using a dye was related to performance, and associate this with teat order. Indigo carmine (5 g/kg) was added to a starter diet between days 12 and 31 (weaning) of lactation. On days 19, 23, 27 and 31, faeces from each piglet were assessed for colouration. Each piglet was categorized as a 'good', 'moderate' or 'small/non' eater of feed. There were no differences in pre-weaning growth rate between categories. Piglets classed as 'good' or 'moderate' eaters in lactation grew fastest (p = 0.009) in the first three days after weaning, but between days 4 and 7, the highest growth rate occurred in 'moderate' eaters. 'Small/ non' eaters grew slower (p < 0.01) between weaning and 28 days after weaning. Piglets drinking milk from anterior teats were heavier at weaning (p < 0.001) and for the first 14 days after weaning (p = 0.104) compared to piglets sucking posterior teats. Data from this study demonstrated that creep feed intake of piglets could qualitatively be assessed using indigo carmine, and that this categorization was related to performance in the immediate post-weaning period.     

Selection of a potential probiotic Lactobacillus strain and subsequent in vivo studies

The probiotic potential of a Lactobacillus strain, isolated from pig faeces, was assessed as a probiotic in piglets. The strain was examined for resistance to pH 2.0, 0.5% oxgall and antibiotics, and antimicrobial activities against enteric pathogenic bacteria. The probiotic strain, L. reuteri BSA131, was administered through the feed to 25 1-month-old Landrace piglets. The piglets were divided into five groups of five piglets each and fed with different diets for 28 days. The daily consumption of L. reuteriBSA131 was assigned into two groups by the concentration of 10⁶ or 10⁸ freeze-dried bacteria. Fecal samples were collected before, during, and after consumption. Lactobacilli and enterobacteria cell counts were determined in the fecal samples. The liveweight gains and feed consumption of the piglets were recorded daily. This study showed that strain BSA 131 enhanced liveweight gains and feed conversion rates in piglets. It also showed a significant increase in lactobacilli cell counts and decreases in enterobacterial numbers in the fecal samples. Strain BSA 131 was considered to be a potential probiotic for piglets, especially after weaning.     

Genetic variation in piglet mortality in outdoor organic production systems

Piglet mortality from farrowing to weaning is a major concern, especially in outdoor organic production systems. This issue might impair animal welfare and generate economic losses for the farmer. In particular, it is difficult to apply management tools that are commonly used for indoor pig production systems to organic or outdoor production systems. Genetics and breeding approaches might be used to improve piglet survival. However, knowledge remains limited on the genetic background underlying survival traits in organic pigs that are born and reared outdoors. Here, we investigated the mortality of piglets from farrowing to weaning in an outdoor organic pig population and suggested genetic strategies to reduce piglet mortality in this production system. The experiment included mortality records of piglets from farrowing to weaning (around 69 days of age). Pedigree-based threshold models were used to analyse the mortality traits of piglets at 0–3 days of age, 4–11 days, and 12 days to weaning. Stillborn piglets were included in the group of piglets that died at 0–3 days of age. We found that the mortality rate from farrowing to weaning was, on average, 19.2%. However, most piglet deaths (79.1%) occurred at 0–11 days of age. As the age of piglets increased, the direct heritability of piglet mortality rose from 0 to 0.04, whereas maternal heritability decreased from 0.03 to a non-significant value. Piglets with higher BW had a lower mortality rate. However, the genetic correlations between maternal effects on piglet mortality and piglet BW were not significant; thus, selection for piglets with higher BW at around 10 days of age, through improving maternal genetics, would not reduce piglet mortality. Piglet mortality increased from sows with increasing number of parities. Crossbreeding also reduced piglet mortality. In conclusion, selection focusing on sow genotype, the use of younger sows, and crossbreeding could contribute to maintain piglet mortality at lower levels in outdoor organic pig production systems.     

Enterococcus faecium EK13--an enterocin a-producing strain with probiotic character and its effect in piglets

The experiment was conducted to determine the effects of the inoculation of the probiotic and enterocin A-producing strain Enterococcus faecium EK13 on selected parameters of metabolic profile, gut microflora, growth, and health in newborn piglets of Slovak White Improved. Piglets for study were divided into two groups: one group (EK13 group, n=8) received strain EK13 per os once daily for 7 days (2ml per piglet, 10(9)CFU/mL of saline buffer). The control group of piglets (n=7) was given placebo-saline buffer. The experiment lasted 14 days. After 7 days, strain EK13 reached 9.8 log(10) CFU/g in faeces of E. faecium EK13 treated piglets while counts of Escherichia coli were significantly lower (P<0.01) than in piglets of the control group. The concentrations of total serum protein, calcium, haemoglobin, haematocrit, red blood cell count and index of phagocytic activity of leukocytes were significantly higher after application of strain EK13. On the other hand, cholesterol was significantly lower in the EK13 group of animals. On day 14, piglets were killed and samples of intestinal contents were taken. Total counts of bacteria in the intestinal contents (jejunum, ileum, caecum, colon) were not significantly influenced. The pH value was significantly lower (P<0.05) only in duodenum of piglets receiving E. faecium EK13. There was a significant higher concentration of lactic acid (P<0.01) and propionic acid in the colon (P<0.001) of the EK13 group. Application of E. faecium EK13 did not influence the daily body weight gain significantly.     

Sow level risk factors for early piglet mortality and crushing in organic outdoor production

Piglet mortality is a major problem in organic pig production affecting both farm economy and animal welfare. Knowledge is scarce on the risk factors of piglet mortality in Danish commercial organic pig production. The objectives of this study were to evaluate season, litter size, parity, sow body condition and stillborn littermates as risk factors for early piglet mortality and crushing of liveborn piglets from parturition until castration at day 3 to 5 postpartum (pp). The study was conducted over a 1-year period in nine commercial Danish organic pig herds practicing outdoor farrowing all year round. Data included recordings on 3393 farrowings with 50 284 liveborn piglets of which 14.8% died before castration. A subset of the dead piglets were collected and necropsied to identify crushed piglets. The average number of liveborn piglets per litter was 14.8 (SD=3.7) and the average time from parturition until castration was 4.1 (SD=1.7) days. A negative binomial regression analysis was used to model the effect of the predictive variables on the early piglet mortality accounting for different time periods from parturition to castration. An increase in maternal body condition score (BCS) and parity significantly increased the risk of dying between parturition and castration. Early mortality was found to be lowest during spring (March to May) and highest during summer (June to August). Being born into a litter with one or more stillborn littermates increased the risk of early mortality. The risk factors for crushing of piglets were evaluated using a logistic analysis. A significant effect of parity and litter size was found where the odds of at least one piglet in a litter with mortality was diagnosed as crushed increased with increasing parity and litter size. In conclusion, being born during summer (June to August), high parity and maternal BCS and stillborn littermates were found to be risk factors for piglet mortality between parturition and castration. In addition, parity and increasing litter size were found to be risk factors for crushing of piglets in litters with mortality.     

The effect of two teeth resection procedures on the welfare of piglets in farrowing crates. Part 1

Teeth resection is a method of controlling the injurious effects of the aggression displayed when newborn piglets fight to establish a teat order. Recent European legislation discourages the practice. The objective of this study was to assess the effect of clipping and grinding piglets’ needle teeth, compared to leaving them intact, on the welfare of piglets in farrowing crates.

Six days pre-partum, 60 sows were assigned to one of three treatments. Litters had their teeth clipped (C), ground (G) or left intact (I) at birth. The time taken to carry out each procedure was recorded. Piglet weights and facial lesions, which were scored according to severity, were recorded on days 1, 4, 11, 18 and 27. Piglet weights were also recorded at birth. Mouth lesions were recorded on days 1, 4 and 27. Instantaneous scan samples of piglet behaviour were carried out for 30 min post-teeth resection procedure (1 min intervals), and for 6 h on days 1, 4, 8, 14, 21 and 26 (5 min intervals). One male and one female piglet per litter were chosen as focal animals and observed for 5 min each post-procedure and for 10 min each twice per day on days 1, 5, 12, 20 and 26. Mortalities were recorded throughout lactation.

Grinding took significantly longer than clipping the teeth or leaving them intact (F = 638.87, P < 0.001). I piglets had higher facial lesion scores than C and G piglets (F = 10.58, P < 0.001). A smaller proportion of piglets in I litters than C and G litters and a smaller proportion of piglets in G litters than C litters had at least one mouth lesion (F = 4.74, P < 0.001). During 30 min post-procedure, I piglets were active on the heatpad in more observations than C and G piglets (F = 3.49, P < 0.05). During 5 min post-procedure C piglets spent longer chomping than I piglets (F = 5.92, P = 0.05). On day 21, I piglets were active in more observations than G piglets (F = 2.11, P < 0.05). On day 26, G piglets were inactive in more observations than C and I piglets (F = 5.02, P < 0.05). On days 14 and 26, C piglets were sleeping in more observations than G piglets (F = 2.87, P = 0.05). There was a tendency for a larger proportion of I than C piglets to die due to overlying (F = 2.68, P = 0.08).

In conclusion, although all three options were associated with welfare problems, grinding can be recommended in preference to clipping or leaving the teeth intact.     

Colonization resistance against Pseudomonas aeruginosa in gnotobiotic mice

Gnotobiotic (GB) mice were colonized with various groups of intestinal bacteria to determine which members of the indigenous flora would exert colonization resistance against Pseudomonas aeruginosa. P. aeruginosa was cultured from the faeces at levels of 10(3)-10(4) cells/g in GB mice inoculated with either the combination of bacteroides and clostridia obtained from conventional (CV) mice or the combination of bacteroides, lactobacilli and clostridia obtained from limited flora mice. The combination of lactobacilli and clostridia from CV mice also did not eliminate P. aeruginosa from GB mice. However, P. aeruginosa was not detected in the faeces of GB mice by 14 days after inoculation with the combination of bacteroides, lactobacilli and clostridia obtained from CV mice. Thus, a complex indigenous flora consisting of bacteroides, lactobacilli and certain clostridia obtained from CV mice but not clostridia obtained from limited flora mice is required to exert complete colonization resistance against P. aeruginosa in GB mice.     

Comparison of agar plate and real-time PCR on enumeration of Lactobacillus, Clostridium perfringens and total anaerobic bacteria in dog faeces

AIMS: To compare agar plate and real-time PCR methods on enumeration of total anaerobic bacteria, Lactobacillus and Clostridium perfringens in dog faeces. METHODS AND RESULTS: Thirty-two faecal specimens from Labrador retriever dogs were used to compare agar plate and real-time PCR enumeration methods for Lactobacillus, C. perfringens and total anaerobic bacteria. Total anaerobic bacteria, C. perfringens and Lactobacillus of faeces were counted (as CFU g(-1) faeces) for 48-h incubation at 37 degrees C in an anaerobic gas chamber on genus-selective media. Total genomic DNA from samples was extracted by the QIAamp DNA stool mini kit. The quantification of DNA (as DNA copy per gram faeces) by real-time PCR was performed with a LightCycler system with the QuantiTect SYBR green PCR kit for PCR amplification. The results indicated that there was a significant correlation between CFU and DNA copy of Lactobacillus (R2 = 0.78, P < 0.01) and total anaerobic bacteria (R2 = 0.21, P < 0.05); but no correlation was found between CFU and DNA copy of C. perfringens. The regression equations for Lactobacillus and total anaerobic bacteria were log(DNA copy) = 0.83 x log(CFU) + 1.43 and log(DNA copy) = 1.62 x log(CFU) - 6.32 respectively. CONCLUSIONS: The real-time PCR method could be used to enumerate Lactobacillus within 2 days when compared with plating method which requires 5-6 days. SIGNIFICANCE AND IMPACT OF THE STUDY: The real-time PCR method and the primer set for Lactobacillus spp. harboured in the dog intestine can be used for rapid enumeration of lactobacilli and monitoring of the faecal Lactobacillus community.     

Reactivity of Limulus amoebocyte lysate towards (1----3)-beta-D-glucans

The structure activity relationship for beta-D-glucans for the gelation of the amoebocyte lysates of the horseshoe crab (Limulus) has been investigated. beta-D-Glucans that had no (1----3) linkages induced little or no gelation. The (1----3)-beta-D-glucans curdlan (unbranched), grifolan (approximately 33% branched), schizophyllan (approximately 33% branched), lentinan (approximately 40% branched). SSG (approximately 50% branched), and OL-2 (approximately 66% branched) induced significant gelation. The optimum concentration for gelation was correlated with the content of branching. Single chain (rather than a triple helix) conformation and higher molecular weight were associated with higher reactivity.