Serum amyloid A levels and alpha 2 and gamma globulins on serum protein electrophoresis in cats exposed to and infected with Leishmania infantum.

BACKGROUND: Dogs are the main reservoir hosts of Leishmania infantum; nevertheless, recent investigations indicate a likely role for cats in the epidemiology of Leishmania infection. Feline leishmaniosis (FeL) remains poorly characterised, partly due to the lack of suitable diagnostic tools. This study aimed to compare serum amyloid A (SAA) levels and serum protein electrophoresis (SPE) profiles (specifically, alpha 2 and gamma globulins) in cats naturally exposed to or infected by L. infantum from southern Italy versus those of healthy controls and versus cats with neoplastic or inflammatory conditions from non-endemic areas. METHODS: Serum or plasma samples from four cohorts of cats were analysed for SAA levels and by SPE: (i) G1: healthy controls from Leishmania-non-endemic regions of Switzerland; (ii) G2: cats pre-diagnosed with neoplastic or inflammatory conditions available from the University of Cambridge sample archive; (iii) G3: L. infantum-seropositive, quantitative (q)PCR-negative cats from southern Italy; (iv) G4: L. infantum-seropositive and qPCR-positive cats from southern Italy. SAA data were assessed for normality and homoscedasticity using the Shapiro-Wilk and Levene's tests, respectively; the Kruskall-Wallis test, followed by Dunn's test with Bonferroni correction were subsequently used to compare SAA serum levels between groups. A weighted generalised linear model with a binomial distribution was used to assess statistically significant differences in the numbers of animals displaying elevated gamma globulins and increased alpha 2 globulins between groups. RESULTS: Overall, 68 samples were analysed (G1: n = 16, G2: n = 20, G3: n = 20, G4: n = 12). Cats suffering from neoplastic and inflammatory conditions (G2 ) showed significantly higher SAA levels than healthy controls (G1) (median values [interquartile range]: G1: 0.00 [0.00-0.00] mg/l versus G2: 0.85 [0.00-49.55] mg/l). G2, G3 and G4 cats showed higher percentages of individuals with increased alpha 2 globulins (percentages ± standard error: G1 = 20.0% ± 10.3, G2 = 80.0% ± 8.9, G3 = 70.0% ± 10.2, G4 = 75.0% ± 12.5) and gamma globulins (G1 = 0.0% ± 0, G2 = 65.0% ± 10.7, G3 = 50.0% ± 11.2, G4 = 58.3% ± 14.2) than healthy control cats (G1). For all three markers, no significant difference between cats within G2, G3 and G4 was recorded. CONCLUSIONS: This study indicates that the proportions of animals with elevated levels of alpha 2 and gamma globulins are significantly higher in cats exposed to and infected with L. infantum. Levels of SAA and alpha 2 and gamma globulins may not be used to differentiate between L. infantum infection or exposure, and neoplastic and/or inflammatory conditions.

Comparison of automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) techniques for analysing the influence of diet on ruminal bacterial diversity.

The objective of this study was to compare the automated ribosomal intergenic spacer analysis (ARISA) and the denaturing gradient gel electrophoresis (DGGE) techniques for analysing the effects of diet on diversity in bacterial pellets isolated from the liquid (liquid-associated bacteria (LAB)) and solid (solid-associated bacteria (SAB)) phase of the rumen. The four experimental diets contained forage to concentrate ratios of 70:30 or 30:70 and had either alfalfa hay or grass hay as forage. Four rumen-fistulated animals (two sheep and two goats) received the diets in a Latin square design. Bacterial pellets (LAB and SAB) were isolated at 2 h post-feeding for DNA extraction and analysed by ARISA and DGGE. The number of peaks in individual samples ranged from 48 to 99 for LAB and from 41 to 95 for SAB with ARISA, and values of DGGE-bands ranged from 27 to 50 for LAB and from 18 to 45 for SAB. The LAB samples from high concentrate-fed animals tended (p < 0.10) to show greater peak numbers and Shannon index values than those isolated from high forage-fed animals with ARISA, but no differences were identified with DGGE. The SAB samples from high concentrate-fed animals had lower (p < 0.05) peak numbers and Shannon index values than those from animals fed high-forage diets with ARISA, but only a trend was noticed for these parameters with DGGE (p < 0.10). The ARISA detected that animals fed alfalfa hay diets showed lower (p < 0.05) SAB diversity than those fed grass hay diets, but no differences were observed with DGGE. No effect of forage type on LAB diversity was detected by any technique. In this study, ARISA detected some changes in ruminal bacterial communities that were not detected by DGGE, and therefore ARISA was considered more appropriate for assessing bacterial diversity of ruminal bacterial pellets. The results highlight the impact of the fingerprinting technique used to draw conclusions on dietary factors affecting bacterial diversity in ruminal bacterial pellets.

Assessment of cecal microbiota, integron occurrence, fermentation responses, and Salmonella frequency in conventionally raised broilers fed a commercial yeast-based prebiotic compound.

Prebiotics are defined as nondigestible food ingredients that can stimulate the growth of one or more beneficial bacteria in the gastrointestinal tract. The Biolex(®) MB40 is a commercial prebiotic that contains mannanoligosaccharides. The aims of this study were to evaluate the effects of prebiotic Biolex(®) MB40 on cecal microbiota of conventionally raised chickens using PCR-based denaturing gradient gel electrophoresis (DGGE) and assessing Salmonella prevalence. Chickens were randomly selected and distributed into three groups; a negative control (NC) and two treatment groups (T1 and T2). The NC group was fed a non-medicated feed, while the treatment groups were fed either T1 or T2, 0.05% antibiotic (BMD50) or 0.2% Biolex(®) MB40 respectively. During the study, cecal contents and bird feed were plated on selective media for Salmonella, yeast and mold prevalence analysis. Ten chickens from each group were randomly selected at 1, 2, 4 and 6 wk and ceca were extracted for DNA isolation for PCR-based DGGE. Also, short-chain fatty acids (SCFAs) were analyzed from collected cecal material by gas chromatography. Only 4.2% of the samples were Salmonella positive. Presence of class 1 integron from cecal material were analyzed by PCR and 97.5% of the cecal samples were positive for integron presence, but no class I integrons were detected in the Salmonella isolates. According to the PCR-based DGGE analysis, the T2 group exhibited a cecal microbial population pattern that was similar to the T1 group prior to wk 4 and the T2 group appeared to be almost identical with the NC group after wk 4 but T2 exhibited less Bacteroides rodentium prior to wk 4. Overall results showed that the commercial prebiotic, MB40 did not lead to a detectable reduction of Salmonella but the general frequency of Salmonella was minimal in all treatments. However, feeding an MB40 supplement did result in similar DGGE band patterns as the T1 group indicating that cecal microbiotia were potentially similar in these 2 groups. Overall, it appears that MB40 (T2) exhibited similar DGGE-cecal population patterns as BMD50 (T1) which suggests that these treatments may have influenced the populations in a comparable fashion.

Effect of purple loosestrife (Lythrum salicaria) diet supplementation in rabbit nutrition on performance, digestibility, health and meat quality.

In this study, 160 Hycole weaned rabbits (35 days old) were randomly divided into four groups of 40. The rabbits were studied throughout a 54-day experimentation period in order to determine the impact of dietary supplementation from herbs composed of 0.2%, 0.4% dry ground Lythrum salicaria leaves (LS) and 0.3% Cunirel(®) (CR; a commercial herb mixture containing LS as the main ingredient) on performance, digestibility, health and meat quality. The basal diet was given to the control group. No significant differences were found in performance, 10 rabbits from each group were selected for evaluation regarding apparent digestibility. The rabbits fed the control diet and the diet with the low level of LS had a higher level of CP digestibility than did the animals that were supplemented with the high LS levels and CR (85.7% and 84.9% v. 84.0% and 84.0%, respectively; P<0.05). The ether extract digestibility was lower in the treatment group with 0.4%LS addition and CR as compared with the control group (52.2% and 54.5% v. 62.6%, respectively; P<0.05). The slaughter process was performed on 89-day-old rabbits to study the carcass characteristics, meat quality, blood parameters, caecal contents and gut histology. The total leukocyte counts in the control animals were lower than they were in the rabbits fed 0.2%, 0.4%LS and CR (4.06 v. 8.25, 8.63 and 8.21×10(9)/l, respectively; P<0.05). For caecal fermentation, the caecal contents of the rabbits fed 0.4% of LS, showed higher concentrations of total volatile fatty acid (VFA; 24.1 v. 18.9 mg/kg dry matter (DM); P<0.05) and acetic acid (18.3 v. 14.4 mg/kg DM; P<0.05), but lower ammonia levels (594 v. 892 mg/kg DM; P<0.05) as compared with the control group. PCR-denaturing gradient gel electrophoresis analyses were performed to evaluate the microbial community in hard faeces, collected at days 35, 42, 49, 56, 70 and 89, whereas the caecal contents were taken after slaughtering. The results demonstrated that between the treatment groups, the similarity of the microbial communities was higher as compared with the control group. Moreover, only age was shown to influence microbiota diversity. In conclusion, the results of this study indicated that supplementation of LS in rabbit diets leads to an increase in the total white blood cells, total VFA and acetic acid concentration, and a decrease in the ammonia levels, as well as the digestibility when CR and high level of LS were supplemented, without causing any adverse effects on other parameters.

Fumonisins affect the intestinal microbial homeostasis in broiler chickens, predisposing to necrotic enteritis.

Fumonisins (FBs) are mycotoxins produced by Fusarium fungi. This study aimed to investigate the effect of these feed contaminants on the intestinal morphology and microbiota composition, and to evaluate whether FBs predispose broilers to necrotic enteritis. One-day-old broiler chicks were divided into a group fed a control diet, and a group fed a FBs contaminated diet (18.6 mg FB1+FB2/kg feed). A significant increase in the plasma sphinganine/sphingosine ratio in the FBs-treated group (0.21 ± 0.016) compared to the control (0.14 ± 0.014) indicated disturbance of the sphingolipid biosynthesis. Furthermore, villus height and crypt depth of the ileum was significantly reduced by FBs. Denaturing gradient gel electrophoresis showed a shift in the microbiota composition in the ileum in the FBs group compared to the control. A reduced presence of low-GC containing operational taxonomic units in ileal digesta of birds exposed to FBs was demonstrated, and identified as a reduced abundance of Candidatus Savagella and Lactobaccilus spp. Quantification of total Clostridium perfringens in these ileal samples, previous to experimental infection, using cpa gene (alpha toxin) quantification by qPCR showed an increase in C. perfringens in chickens fed a FBs contaminated diet compared to control (7.5 ± 0.30 versus 6.3 ± 0.24 log10 copies/g intestinal content). After C. perfringens challenge, a higher percentage of birds developed subclinical necrotic enteritis in the group fed a FBs contaminated diet as compared to the control (44.9 ± 2.22% versus 29.8 ± 5.46%).

Identification of intestinal bacterial flora in Rhipicephalus microplus ticks by conventional methods and PCR-DGGE analysis.

In this study, we have analyzed the intestinal microbial flora associated with Rhipicephalus microplus ticks using both culture-dependent and independent methods based on PCR and denaturing gradient gel electrophoresis (PCR-DGGE). The R. microplus ticks were collected from cattle and goats in Jiangxi, Hunan and Guizhou Provinces of China. Three distinct strains of bacteria were isolated using culture-dependent methods: Staphylococcus simulans, Bacillus subtilis and Bacillus flexus strain. Nineteen distinct DGGE bands were found using PCR-DGGE analysis, and their search for identity shows that they belonged to Rickettsiaceae, Xanthomonadaceae, Coxiella sp., Ehrlichia sp., Pseudomonas sp., Ehrlichia sp., Orphnebius sp., Rickettsia peacockii, Bacillus flexus. Rickettsia peacockii and Coxiella genus were the dominant strain of the R. microplus ticks from cattle, Pseudomonas sp. and B. flexus strain were the most common species in all tick samples from goats. Ehrlichia canis were detected only in R. microplus ticks from Yongshun area in Hunan Province. The results indicate that the intestinal microbial diversity of R. microplus ticks was influenced by tick hosts and local differences in the sampling location and these two aspects may affect transmission of pathogen to humans and animals.

Effect of dietary oxidized konjac glucomannan on Schizothorax prenanti growth performance, body composition, intestinal morphology and intestinal microflora.

The aim of the present study was to examine the effect of oxidized konjac glucomannan (OKGM) on Schizothorax prenanti growth performance, body composition, intestinal morphology and intestinal microflora. Fish were fed a basal diet or basal diet plus 4.0, 8.0, 16.0 and 32.0 g kg(-1) OKGM for 60 days. The results indicated that WGR and SGR were significantly higher in fish fed 8.0 and 16.0 g kg(-1) OKGM diets (P < 0.05) than those in fish fed basal diet, and PER was significantly higher and FCR was significantly lower in fish fed 16.0 g kg(-1) OKGM diet (P < 0.05). The content of body protein, lipid and moisture was affected by the OKGM diets. The light and electron microscopy demonstrated that intestinal morphology of fish fed 8.0 and 16.0 g kg(-1) OKGM diet was better (P < 0.05) than the control group, including mucosa fold height, mucosal epithelial height, submucosa height, longitudinal muscularis thickness and circular muscularis thickness. Compared with the control group, fish fed 32.0 g kg(-1) OKGM diet showed significantly lower goblet cell number in anterior intestine (P < 0.05). Furthermore, intestinal microflora was analyzed by PCR-DGGE, and the results showed that OKGM diets also significantly modulated the intestinal microflora of fish (P < 0.05). The study clearly demonstrates that OKGM could enhance the growth performance, improve intestinal morphology and modulate intestinal microflora of S. prenanti, and the optimal dietary OKGM levels was suggested to be 16.0 g kg(-1).

Alterations of digestive enzyme activities, intestinal morphology and microbiota in juvenile paddlefish, Polyodon spathula, fed dietary probiotics.

The effects of dietary supplementation of probiotics on digestive enzymes activities, intestinal morphology and microbiota in juvenile paddlefish (Polyodon spathula) were studied. A total of 400 fish were reared in two cages and fed with a basal diet (control group, CG) or diet supplemented with commercial probiotics (treatment group, TG) for 80 days. Enzymes activities analysis indicated that protease and α-amylase activities increased (P < 0.01 or P < 0.05) in TG. Light microscopy observation demonstrated the decrease of wall thickness and muscularis thickness in foregut (P < 0.01), the increase of those in hindgut (P < 0.05), the increase of folds height in foregut (P < 0.01) and midgut in TG (P < 0.05). DGGE results of PCR-amplified 16S rRNA confirmed that the richness and diversity of intestinal microbial species increased in TG. The similarity between the commercial bacteria product and intestinal microbiota of TG were higher than the microbiota from CG. The quantities of bacterium, Firmicutes, Proteobacteria, Bacteroidetes, Fusobacteria, present an increasing trend from foregut to hindgut both in two groups. To our knowledge, this is the first in vivo study to reveal the effect of dietary probiotics on intestinal digestive enzymes activities, morphology and microbiota in paddlefish.

First isolation of Mycoplasma iowae in grey partridge flocks.

Mycoplasma iowae, an occasional pathogen of turkeys, was isolated for the first time from captive grey partridges (Perdix perdix). Clinical signs including respiratory and intestinal disorder were seen in birds of all ages but mainly in those kept housed during rearing. Mortality rates averaged over 20% during the year. Treatment with antibiotics and antiparasitic drugs produced only a transient improvement in condition. The gross pathology findings included poor body growth, lack of development of the breast muscles, abnormalities in the keel development, and bone fragility. Some birds showed infraorbital sinusitis with serous or fibrinous exudates and catarrhal tracheitis, while others presented serofibrinous airsacculitis and splenomegaly. Laboratory investigations revealed pure cultures of M. iowae in the gut as well as sinus and air sacs. While other organisms such as coccidia, Trichomonas, Escherichia coli, Clostridium perfringens, and Aspergillus spp. were detected, the similarity of the disease with that seen in turkeys infected with M. iowae strongly suggests that this mycoplasma may be the primary pathogen here. The presence of M. iowae in game birds commonly released into the wild could have serious implications particularly in areas where industrial poultry farms are concentrated.

Effect of Origanum chemotypes on broiler intestinal bacteria.

Essential oils have been proposed as alternatives to antibiotic use in food animal production. This study evaluated 3 chemotypes of the Origanum genus, containing varying amounts of secondary metabolites carvacrol, thymol, and sabinene, in the broiler chicken diet. Aerial parts of Origanum vulgare L. (OL), O. vulgare L. ssp. hirtum (OH), and O. majorana (OM) were collected from a greenhouse located in the high altitude Sabana de Bogotá (Savanna of Bogotá) and O. vulgare L. ssp. hirtum (OG) produced and ground in Greece. Oregano essential oils (OEO) from these plants were obtained by steam distillation and analyzed by gas chromatography coupled to a mass spectrometer. Six treatments were evaluated: 200 mg/kg of OEO from OH, OL, and OM, 50 mg/kg of OEO from OG, 500 mg/kg of chlortetracycline, and without additives. Broiler chicks were maintained at 2,600 m above sea level, placed in brooder cages under a completely randomized design. Template DNA was isolated from duodenal, jejunal, ileal, and cecal contents in each group and bacterial 16S rDNA patterns were analyzed by denaturing gradient gel electrophoresis. Dendrograms of denaturing gradient gel electrophoresis band patterns revealed 2 main clusters, OEO-treated chicks and nontreated control chicks, in each intestinal segment. Band patterns from different gut compartments revealed major bacterial population shifts in the foregut (duodenum, jejunum, and ileum) compared with the hindgut (cecum and colon) at all ages evaluated (P < 0.05). The OEO groups showed less shift (62.7% similarity coefficient) between these 2 compartments versus the control groups (53.7% similarity coefficient). A reduction of 59% in mortality from ascites was seen in additive-supplemented groups compared with the control group. This study represents the first work to evaluate the effects of the 3 main chemotypes of Origanum genus in broilers.

Studies into the prevalence of Mycoplasma species in small ruminants in Benue State, North-central Nigeria.

The indicative prevalence of respiratory Mycoplasma species in small ruminants (SR) was determined in North-central Nigeria. Nasal swabs from 172 sheep and 336 goats from the Northeast, Northwest and South Senatorial Districts of Benue State were examined. Initial Mycoplasma isolation used Mycoplasma culture techniques followed by digitonin sensitivity testing. Species identification was done using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). Overall, Mycoplasma organisms were isolated from 131 (25.8 %) of the 508 SR examined. Prevalence rates of 18.1 and 29.8 % were recorded for sheep and goats, respectively. A total of 135 isolates of Mycoplasma belonging to three different species were identified: Mycoplasma ovipneumoniae (127), Mycoplasma arginini (7) and Mycoplasma mycoides subspecies capri (1). More than one Mycoplasma species were detected in four (3.1 %) of the 131 confirmed Mycoplasma positive cultures. Mycoplasma was isolated from 16.2 and 29.1 % of animals with and without respiratory signs, respectively. The high isolation rate of mycoplasmas in apparently healthy and clinically sick sheep and goats in this study indicates a carrier status in these SR which may constitute a serious problem in disease control.

Bona fide evidence for natural vertical transmission of infectious salmon anemia virus in freshwater brood stocks of farmed Atlantic salmon (Salmo salar) in Southern Chile.

UNLABELLED: Infectious salmon anemia (ISA) is a severe disease that affects farmed Atlantic salmon (Salmo salar), causing outbreaks in seawater in most salmon-producing countries worldwide, with particular aggressiveness in southern Chile. The etiological agent of this disease is a virus belonging to the Orthomyxoviridae family, named infectious salmon anemia virus (ISAV). Although it has been suggested that this virus can be vertically transmitted, even in freshwater, there is a lack of compelling experimental evidence to confirm this. Here we demonstrate significant putative viral loads in the ovarian fluid as well as in the eggs of two brood stock female adult specimens that harbored the virus systemically but without clinical signs. The target virus corresponded to a highly polymorphic region 3 (HPR-3) variant, which is known to be virulent in seawater and responsible for recent and past outbreaks of this disease in Chile. Additionally, the virus recovered from the fluid as well as from the interior of the eggs was fully infective to a susceptible fish cell line. To our knowledge, this is the first robust evidence demonstrating mother-to-offspring vertical transmission of the infective virus on the one hand and the asymptomatic transmission of a virulent form of the virus in freshwater fish on the other hand. IMPORTANCE: The robustness of the data presented here will contribute to a better understanding of the biology of the virus but most importantly will constitute a key management tool in the control of an aggressive agent constantly threatening the sustainability of the global salmon industry.

Technical note: Comparison of automated ribosomal intergenic spacer analysis and denaturing gradient gel electrophoresis to assess bacterial diversity in the rumen of sheep.

The aim of this study was to compare automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) techniques to assess bacterial diversity in the rumen of sheep. Sheep were fed 2 diets with 70% of either alfalfa hay or grass hay, and the solid (SOL) and liquid (LIQ) phases of the rumen were sampled immediately before feeding (0 h) and at 4 and 8 h postfeeding. Both techniques detected similar differences between forages, with alfalfa hay promoting greater (P < 0.05) bacterial diversity than grass hay. In contrast, whereas ARISA analysis showed a decrease (P < 0.05) of bacterial diversity in SOL at 4 h postfeeding compared with 0 and 8 h samplings, no variations (P > 0.05) over the postfeeding period were detected by DGGE. The ARISA technique showed lower (P < 0.05) bacterial diversity in SOL than in LIQ samples at 4 h postfeeding, but no differences (P > 0.05) in bacterial diversity between both rumen phases were detected by DGGE. Under the conditions of this study, the DGGE was not sensitive enough to detect some changes in ruminal bacterial communities, and therefore ARISA was considered more accurate for assessing bacterial diversity of ruminal samples. The results highlight the influence of the fingerprinting technique used to draw conclusions on factors affecting ruminal bacterial diversity.

Does anaerobic bacterial antibiosis decrease fungal diversity in oral necrobacillosis disease?

Oral necrobacillosis (ON) is a model polymicrobial disease that affects macropods in captivity and livestock. Several studies in humans and animals have focused mainly on the bacterial etiology of this disease with little or no information on the role/association of fungi with ON. Using a Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) assay and statistical analysis of the fungal community structure in healthy and disease groups, a reduction in the species diversity and drastic reduction (>1000 fold) in the fungal population in wallabies with ON was observed. Furthermore, an in vitro assay revealed a potential anaerobic-bacteria antibiosis mechanism in the observed decrease in fungal population in ON and a synergistic bacterial-fungal interaction in wallabies with healthy oral status. This study contributes to our knowledge of the fungal community structure associated with ON and forms the basis for an investigation at an epidemiological scale in order to exploit the clinical potentials of these findings.

The oral microbial community of gingivitis and lumpy jaw in captive macropods.

Gingivitis and lumpy jaw are diseases of polymicrobial aetiology. Although Fusobacterium necrophorum has been associated with these diseases in macropods, little is known about other organisms associated with these diseases in this animal species. PCR-DGGE analysis revealed the potential pathogens associated with gingivitis and lumpy jaw in macropods. PCR-DGGE profile comparison between the healthy and disease groups indicated a shift in the oral bacterial community structures with similarity coefficients of 48% and 35% for gingivitis and lumpy jaw respectively. Moreover, gingivitis was associated with increase in bacterial diversity (Shannon index = 2.87; PL curve = 45%) while lumpy jaw resulted in a decline in bacterial diversity (Shannon index = 2.47; PL curve = 74%). This study suggest that the establishment of gingivitis and lumpy jaw diseases follows the ecological plaque hypothesis. This forms the basis for an expanded investigation in an epidemiological scale and suggests the need for the appropriate choice of antimicrobial agent(s) and for the effective management and control of polymicrobial diseases.

Lactobacillus planarum subsp. plantarum JCM 1149 vs. Aeromonas hydrophila NJ-1 in the anterior intestine and posterior intestine of hybrid tilapia Oreochromis niloticus ♀ × Oreochromis aureus ♂: an ex vivo study.

To investigate the ex vivo interactions of probiotic-pathogen-host in warm-water fish, hybrid tilapia (Oreochromis niloticus♀ × Oreochromis aureus♂) were sacrificed to isolate anterior and posterior intestine for incubation with phosphate-buffered saline (PBS; pH 7.2) as the control, Lactobacillus plantarum JCM 1149 at 1.0 × 10(9) CFU/ml, Aeromonas hydrophila NJ-1 at 1.0 × 10(8) CFU/ml, or the both combination. Denaturing Gradient Gel Electrophoresis (DGGE) fingerprint and consequent sequence analysis confirmed anterior intestine sac was more prone to the colonization of L. plantarum JCM 1149 and A. hydrophila NJ-1 than the posterior part. L. plantarum JCM 1149 and A. hydrophila NJ-1 inhibited the population each other in anterior or posterior sac, indicating their competition for the colonization. The induced expression of HSP70, IL-1ß and TNF-α in the anterior sac by the addition of L. plantarum JCM 1149 or A. hydrophila NJ-1 demonstrated the activity and a local immune response of ex vivo anterior sac. Compared with posterior intestine, higher population colonization and more sensitive immune response of anterior sac indicated differential patterns for the probiotic-pathogen-host interactions. Scanning electronic microscopy (SEM) observation showed that pathogen A. hydrophila NJ-1 damaged the anterior intestine, which was alleviated by the pretreatment of L. plantarum JCM 1149, showing its probiotic effect.

Changes in the rumen epimural bacterial diversity of beef cattle as affected by diet and induced ruminal acidosis.

Little is known about the nature of the rumen epithelial adherent (epimural) microbiome in cattle fed different diets. Using denaturing gradient gel electrophoresis (DGGE), quantitative real-time PCR (qPCR), and pyrosequencing of the V3 hypervariable coding region of 16S rRNA, epimural bacterial communities of 8 cattle were profiled during the transition from a forage to a high-concentrate diet, during acidosis, and after recovery. A total of 153,621 high-quality gene sequences were obtained, with populations exhibiting less taxonomic variability among individuals than across diets. The bacterial community composition exhibited clustering (P < 0.03) by diet, with only 14 genera, representing >1% of the rumen epimural population, differing (P ≤ 0.05) among diets. During acidosis, levels of Atopobium, Desulfocurvus, Fervidicola, Lactobacillus, and Olsenella increased, while during the recovery, Desulfocurvus, Lactobacillus, and Olsenella reverted to levels similar to those with the high-grain diet and Sharpea and Succinivibrio reverted to levels similar to those with the forage diet. The relative abundances of bacterial populations changed during diet transition for all qPCR targets except Streptococcus spp. Less than 5% of total operational taxonomic units (OTUs) identified exhibited significant variability across diets. Based on DGGE, the community structures of epithelial populations differed (P ≤ 0.10); segregation was most prominent for the mixed forage diet versus the grain, acidotic challenge, and recovery diets. Atopobium, cc142, Lactobacillus, Olsenella, RC39, Sharpea, Solobacterium, Succiniclasticum, and Syntrophococcus were particularly prevalent during acidosis. Determining the metabolic roles of these key genera in the rumens of cattle fed high-grain diets could define a clinical microbial profile associated with ruminal acidosis.

The effects of liquid versus spray-dried Laminaria digitata extract on selected bacterial groups in the piglet gastrointestinal tract (GIT) microbiota.

In this study, the effects of supplementing animal feed with a liquid and spray-dried fucoidan and laminarin extract, derived from the seaweed Laminaria digitata on the porcine gastrointestinal microbiota, specifically the communities of Lactobacillus, Bifidobacterium and enterobacteria were evaluated. Twenty four piglets were fed one of three diets over a 21-day period to determine the effect that each had on the bacterial communities. The dietary treatments were as follows; (1) control diet, (2) control diet plus spray-dried formulation of laminarin fucoidan (L/F-SD) extract, (3) control diet plus a liquid formulation of (L/F-WS) extract. Control diet consisted of wheat, soya bean meal, soya oil and a vitamin and mineral mixture. The L/F-SD and L/F-WS supplemented diets had equal proportion of 500 ppm laminarin and fucoidan. At the end of the 21 day feeding period all animals were sacrificed and samples were collected from the ileum, caecum and colon. Counts were determined for Lactobacillus, Bifidobacterium and enterobacteria. Plate count analysis revealed that the L/F-SD diet caused a statistically significant 1.5 log and 2 log increases in the Lactobacillus and Bifidobacterium counts of ileum samples respectively. A greater difference was observed with the L/F-WS diet in that Lactobacillus and Bifidobacterium increased by 2 log and 3 log respectively. Alterations in the Lactobacillus species composition of the gastrointestinal tract (GIT) were analysed using specific PCR - denaturing gradient gel electrophoresis (PCR-DGGE). The DGGE profiles indicated that Lactobacillus species richness decreased along the gastrointestinal tract i.e. the number of dominant species detected in the colon was less than those detected in the ileum and caecum irrespective of the diet consumed. Consumption of both the L/F-SD and L/F-WS diets resulted in a richer Lactobacillus species composition in the ileum, with the L/F-SD diet being associated the emergence of Lactobacillus agilis in the colon. The study indicated that the L/F-WS extract was superior to the L/F-SD extract in increasing the titre of beneficial bacteria in the gastrointestinal tract (GIT).

Analysis of bacterial community shifts in the gastrointestinal tract of pigs fed diets supplemented with ß-glucan from Laminaria digitata, Laminaria hyperborea and Saccharomyces cerevisiae.

This study was designed to evaluate the effects of algal and yeast ß-glucans on the porcine gastrointestinal microbiota, specifically the community of Lactobacillus, Bifidobacterium and coliforms. A total of 48 pigs were fed four diets over a 28-day period to determine the effect that each had on these communities. The control diet consisted of wheat and soya bean meal. The remaining three diets contained wheat and soya bean meal supplemented with ß-glucan at 250 g/tonne from Laminaria digitata, Laminaria hyperborea or Saccharomyces cerevisiae. Faecal samples were collected from animals before feeding each diet and after the feeding period. The animals were slaughtered the following day and samples were collected from the stomach, ileum, caecum, proximal colon and distal colon. Alterations in Lactobacillus in the gastrointestinal tract (GIT) were analysed using denaturing gradient gel electrophoresis (DGGE) profiles generated by group-specific 16S rRNA gene PCR amplicons. Plate count analysis was also performed to quantify total coliforms. DGGE profiles indicated that all ß-glucan diets provoked the emergence of a richer community of Lactobacillus. The richest community of lactobacilli emerged after feeding L. digitata (LD ß-glucan). Plate count analysis revealed that the L. hyperborea (LH ß-glucan) diet had a statistically significant effect on the coliform counts in the proximal colon in comparison with the control diet. ß-glucan from L. digitata and S. cerevisiae also generally reduced coliforms but to a lesser extent. Nevertheless, the ß-glucan diets did not significantly reduce levels of Lactobacillus or Bifidobacterium. DGGE analysis of GIT samples indicated that the three ß-glucan diets generally promoted the establishment of a more varied range of Lactobacillus species in the caecum, proximal and distal colon. The LH ß-glucan had the most profound reducing effect on coliform counts when compared with the control diet and diets supplemented with L. digitata and S. cerevisiae ß-glucans.

Effect of dam parity on litter performance, transfer of passive immunity, and progeny microbial ecology.

Litter performance and progeny health status may be decreased in progeny derived from primiparous sows but improve with increasing parity. The objective was to evaluate litter performance, the production and passive transfer of Ig, and fecal microbial populations in progeny derived from first parity (P1) compared with fourth parity (P4) dams. Litter performance was recorded for P1 (n = 19) and P4 (n = 24) dams including number of pigs/litter (total born, born live, stillbirths, mummified fetuses, prewean mortality, and pigs weaned) and average litter and piglet BW at birth (d 0), d 7, d 14, and at weaning (average d 19). Blood samples were collected from all dams on d 90 and 114 of gestation and d 0 of lactation. Colostrum and milk samples were collected from each dam on d 0, 7, and 14 of lactation for quantification of IgG and IgA. Blood and fecal samples were collected from each litter (n = 6 pigs/litter) on d 1, 7, and 14 after parturition. Circulating IgG and IgA concentrations were quantified in all blood samples. Denaturing gradient gel electrophoresis (DGGE) was used to characterize similarity and diversity of fecal microbes among progeny. Progeny of P1 dams had decreased average litter BW at d 7 (25.7 vs. 30.0 kg; P < 0.03) and decreased average piglet BW throughout the experiment (d 0, 7, 14, and 19; P < 0.001) compared with P4 progeny. No parity × day interactions were observed with respect to immunoglobulin or microbial analyses. Concentrations of IgA tended to be greater (P = 0.09) in samples of colostrum and milk obtained from P4 compared with P1 dams. Serum IgG concentrations were greater (P < 0.02) in P4 progeny compared with P1 progeny. Results of DGGE revealed that P1 progeny had increased (P < 0.001) microbial similarity on d 7 and decreased (P < 0.03) microbial similarity on d 14 compared with P4 progeny. Progeny of P1 dams tended (P = 0.07) to have a greater Shannon's diversity index compared with P4 progeny on d 1, and P1 progeny had a greater (P < 0.03) Shannon's diversity index compared with P4 progeny on d 7. Litter performance, passive transfer of immunity, and progeny microbial ecology were affected by dam parity.