Maternal and/or post-weaning supplementation with Bacillus altitudinis spores modulates the microbial composition of colostrum, digesta and faeces in pigs.

This study examined the effects of maternal and/or post-weaning Bacillus altitudinis supplementation on the microbiota in sow colostrum and faeces, and offspring digesta and faeces. Sows (n = 12/group) were assigned to: (1) standard diet (CON), or (2) CON supplemented with probiotic B. altitudinis spores (PRO) from day (d)100 of gestation to weaning (d26 of lactation). At weaning, offspring were assigned to CON or PRO for 28d, resulting in: (1) CON/CON, (2) CON/PRO, (3) PRO/CON, and (4) PRO/PRO, after which all received CON. Samples were collected from sows and selected offspring (n = 10/group) for 16S rRNA gene sequencing. Rothia was more abundant in PRO sow colostrum. Sow faeces were not impacted but differences were identified in offspring faeces and digesta. Most were in the ileal digesta between PRO/CON and CON/CON on d8 post-weaning; i.e. Bacteroidota, Alloprevotella, Prevotella, Prevotellaceae, Turicibacter, Catenibacterium and Blautia were more abundant in PRO/CON, with Firmicutes and Blautia more abundant in PRO/PRO compared with CON/CON. Lactobacillus was more abundant in PRO/CON faeces on d118 post-weaning. This increased abundance of polysaccharide-fermenters (Prevotella, Alloprevotella, Prevotellaceae), butyrate-producers (Blautia) and Lactobacillus likely contributed to previously reported improvements in growth performance. Overall, maternal, rather than post-weaning, probiotic supplementation had the greatest impact on intestinal microbiota.

Maternal supplementation with Bacillus altitudinis spores improves porcine offspring growth performance and carcass weight.

The objective of this study was to evaluate the effect of feeding Bacillus altitudinis spores to sows and/or offspring on growth and health indicators. On day (D) 100 of gestation, twenty-four sows were selected and grouped as: control (CON), fed with a standard diet; and probiotic (PRO), fed the standard diet supplemented with B. altitudinis WIT588 spores from D100 of gestation until weaning. Offspring (n 144) from each of the two sow treatments were assigned to either a CON (no probiotic) or PRO (B. altitudinis-supplemented) treatment for 28 d post-weaning (pw), resulting in four treatment groups: (1) CON/CON, non-probiotic-supplemented sow/non-probiotic-supplemented piglet; (2) CON/PRO, non-probiotic-supplemented sow/probiotic-supplemented piglet; (3) PRO/CON, probiotic-supplemented sow/non-probiotic-supplemented piglet and (4) PRO/PRO, probiotic-supplemented sow/probiotic-supplemented piglet. B. altitudinis WIT588 was detected in the faeces of probiotic-supplemented sows and their piglets, and in the faeces and intestine of probiotic-supplemented piglets. Colostrum from PRO sows had higher total solids (P = 0·02), protein (P = 0·04) and true protein (P = 0·05), and lower lactose (P < 0·01) than colostrum from CON sows. Maternal treatment improved offspring feed conversion ratio at D0-14 pw (P < 0·001) and increased offspring body weight at D105 and D127 pw (P = 0·01), carcass weight (P = 0·05) and kill-out percentage (P < 0·01). It also increased small intestinal absorptive capacity and impacted the haematological profile of sows and progeny. There was little impact of pw treatment on any of the parameters measured. Overall, the lifetime growth benefits in the offspring of B. altitudinis-supplemented sows offer considerable economic advantages for pig producers in search of alternatives to in-feed antibiotics/zinc oxide.

Intestinal microbiota modulation and improved growth in pigs with post-weaning antibiotic and ZnO supplementation but only subtle microbiota effects with Bacillus altitudinis.

The objective was to evaluate the effect of dietary Bacillus altitudinis spore supplementation during day (D)0-28 post-weaning (PW) and/or D29-56 PW compared with antibiotic and zinc oxide (AB + ZnO) supplementation on pig growth and gut microbiota. Eighty piglets were selected at weaning and randomly assigned to one of five dietary treatments: (1) negative control (Con/Con); (2) probiotic spores from D29-56 PW (Con/Pro); (3) probiotic spores from D0-28 PW (Pro/Con); (4) probiotic spores from D0-56 PW (Pro/Pro) and (5) AB + ZnO from D0-28 PW. Overall, compared with the AB + ZnO group, the Pro/Con group had lower body weight, average daily gain and feed intake and the Pro/Pro group tended to have lower daily gain and feed intake. However, none of these parameters differed between any of the probiotic-treated groups and the Con/Con group. Overall, AB + ZnO-supplemented pigs had higher Bacteroidaceae and Prevotellaceae and lower Lactobacillaceae and Spirochaetaceae abundance compared to the Con/Con group, which may help to explain improvements in growth between D15-28 PW. The butyrate-producing genera Agathobacter, Faecalibacterium and Roseburia were more abundant in the Pro/Con group compared with the Con/Con group on D35 PW. Thus, whilst supplementation with B. altitudinis did not enhance pig growth performance, it did have a subtle, albeit potentially beneficial, impact on the intestinal microbiota.

Recent advances in fermented feeds towards improved broiler chicken performance, gastrointestinal tract microecology and immune responses: A review.

Previously, fermentation has been associated with methods that improve the nutritional value of unconventional feed ingredients for broilers. In recent decades, the fermentation process has been employed to produce functional feeds that have the potential to improve broiler gastrointestinal tract microecology, health and production performance. Some of the functional ingredients found in fermented feed include lactic acid bacteria (LAB), lactic acid and other organic acids, and appear to play major roles in determining the beneficial effects of fermented feed on broiler gut health and performance. Unlike the pig, the available literature on broiler fermented feed is still rather limited. This review describes recent advances in the use of fermented feed (on the basis of conventional and unconventional feed ingredients) in broilers. Similarly, this review also shows that additional research is necessary to exploit fermented feed as a viable food source in broiler nutrition.

Influence of feeding crimped kernel maize silage on the course of subclinical necrotic enteritis in a broiler disease model.

This experiment was carried out with 375 male broilers (Ross 308) from days 1 to 28 to evaluate the influence of crimped kernel maize silage (CKMS) on the manifestation of subclinical necrotic enteritis, microbiota counts, organic acid production and relative weights of gastrointestinal segments. A necrotic enteritis disease model was applied. Birds were allocated into 3 different dietary treatments: a maize-based feed (MBF, control diet), and 2 diets supplemented with 15% (CKMS15) or 30% (CKMS30) of crimped ensiled kernel maize. The disease model involved a 10-time overdose of an attenuated live vaccine against coccidiosis given orally on day 17, followed by oral inoculation of Clostridium perfringens Type A (S48, 108 to 109 bacteria/bird) twice daily on days 18, 19, 20 and 21. Scoring of intestinal lesions was performed on days 22, 23, 25 and 28. Ileal and caecal digesta samples were collected for the quantification of selected bacterial groups and organic acids. The results showed that there was no effect of dietary treatments on small intestinal lesion scores (P > 0.05). Lesions scores peaked on days 23 and 25 and decreased again on day 28 (P = 0.001). No effect of age on microbiota counts was observed, but feeding of CKMS30 reduced the number of coliforms in ileal contents (P = 0.01). Dietary treatments did not affect organic acid concentrations in ileum and caeca, but there was an effect of age; butyric acid was higher on days 22, 23 and 25 than on day 28 (P = 0.04). Acetic acid and propionic acid concentrations in caeca were the highest on days 22 and 28 but the lowest on days 23 and 25. Relative gizzard and caeca weights were increased, and relative ileum weights were decreased when birds were fed CKMS30 (P < 0.05). In conclusion, the inclusion of CKMS in broiler diets had no effects on the course of necrotic enteritis but had potential benefits in terms of inhibition of potentially harmful microorganisms.

The influence of feeding crimped kernel maize silage on growth performance and intestinal colonization with Campylobacter jejuni of broilers.

An infection trial and a production trial over 35 days were conducted in parallel to study the influence of feeding crimped kernel maize silage (CKMS) on the intestinal Campylobacter jejuni colonization and broiler performance, respectively. The CKMS was used at dietary inclusion levels of 15% and 30% in maize-based diets. Broilers were orally inoculated with 2 × 10(5) log cfu/ml C. jejuni on day 14. Four birds from each pen were randomly selected and killed by cervical dislocation on days 3, 6, 9, 14 and 21 post infection and intestinal contents from ileum, caeca and rectum as well as liver samples were taken. Body weight and feed consumption of broilers were registered on days 13, 22 and 35. On day 35, litter dry matter (DM) was measured and the condition of the foot pads was evaluated. There was no significant effect of CKMS on the colonization of C. jejuni. Body weight of the broilers supplemented with 15% CKMS was comparable with the control maize-based feed, whereas addition of 30% CKMS reduced broiler body weight (P < 0.001). However, DM intake and feed conversion ratio were the same in all three dietary treatments. Furthermore, the foot pad condition of broilers significantly improved with the inclusion of CKMS on broiler diets as a result of a higher DM content in the litter material. It is concluded that CKMS did not influence intestinal Campylobacter colonization, but improved the foot pad health of broilers.

Bacterial Succession in the Broiler Gastrointestinal Tract.

A feeding trial was performed with broilers receiving a diet of wheat-based feed (WBF), maize-based feed (MBF), or maize-based concentrates supplemented with 15% or 30% crimped kernel maize silage (CKMS-15 or CKMS-30, respectively). The aim of the study was to investigate the bacterial community compositions of the crop, gizzard, ileum, and cecum contents in relation to the feeding strategy and age (8, 15, 22, 25, 29, or 36 days). Among the four dietary treatments, bacterial diversity was analyzed for MBF and CKMS-30 by 454 pyrosequencing of the 16S rRNA gene. Since the diets had no significant influence on bacterial diversity, data were pooled for downstream analysis. With increasing age, a clear succession of bacterial communities and increased bacterial diversity were observed.Lactobacillaceae(belonging mainly to the genus Lactobacillus) represented most of the Firmicutesat all ages and in all segments of the gut except the cecum. The development of a "mature" microbiota in broilers occurred during the period from days 15 to 22. Striking increases in the relative abundances of Lactobacillus salivarius(17 to 36%) and clostridia (11 to 18%), and a concomitant decrease in the relative abundance of Lactobacillus reuteri, were found in the ileum after day 15. The concentration of deconjugated bile salts increased in association with the increased populations of L. salivarius and clostridia. Both L. salivarius and clostridia deconjugate bile acids, and increases in the abundances of these bacteria might be associated with growth reduction and gastrointestinal (GI) disorders occurring in the critical period of broiler life between days 20 and 30.

Multiple Origins of Mutations in the mdr1 Gene--A Putative Marker of Chloroquine Resistance in P. vivax.

BACKGROUND: Chloroquine combined with primaquine has been the recommended antimalarial treatment of Plasmodium vivax malaria infections for six decades but the efficacy of this treatment regimen is threatened by chloroquine resistance (CQR). Single nucleotide polymorphisms (SNPs) in the multidrug resistance gene, Pvmdr1 are putative determinants of CQR but the extent of their emergence at population level remains to be explored. OBJECTIVE: In this study we describe the prevalence of SNPs in the Pvmdr1 among samples collected in seven P. vivax endemic countries and we looked for molecular evidence of drug selection by characterising polymorphism at microsatellite (MS) loci flanking the Pvmdr1 gene. METHODS: We examined the prevalence of SNPs in the Pvmdr1 gene among 267 samples collected from Pakistan, Afghanistan, Sri Lanka, Nepal, Sudan, São Tomé and Ecuador. We measured and diversity in four microsatellite (MS) markers flanking the Pvmdr1 gene to look evidence of selection on mutant alleles. RESULTS: SNP polymorphism in the Pvmdr1 gene was largely confined to codons T958M, Y976F and F1076L. Only 2.4% of samples were wildtype at all three codons (TYF, n = 5), 13.3% (n = 28) of the samples were single mutant MYF, 63.0% of samples (n = 133) were double mutant MYL, and 21.3% (n = 45) were triple mutant MFL. Clear geographic differences in the prevalence of these Pvmdr mutation combinations were observed. Significant linkage disequilibrium (LD) between Pvmdr1 and MS alleles was found in populations sampled in Ecuador, Nepal and Sri Lanka, while significant LD between Pvmdr1 and the combined 4 MS locus haplotype was only seen in Ecuador and Sri Lanka. When combining the 5 loci, high level diversity, measured as expected heterozygosity (He), was seen in the complete sample set (He = 0.99), while He estimates for individual loci ranged from 0.00-0.93. Although Pvmdr1 haplotypes were not consistently associated with specific flanking MS alleles, there was significant differentiation between geographic sites which could indicate directional selection through local drug pressure. CONCLUSIONS: Our observations suggest that Pvmdr1 mutations emerged independently on multiple occasions even within the same population. In Sri Lanka population analysis at multiple sites showed evidence of local selection and geographical dispersal of Pvmdr1 mutations between sites.

Global and local genetic diversity at two microsatellite loci in Plasmodium vivax parasites from Asia, Africa and South America.

BACKGROUND: Even though Plasmodium vivax has the widest worldwide distribution of the human malaria species and imposes a serious impact on global public health, the investigation of genetic diversity in this species has been limited in comparison to Plasmodium falciparum. Markers of genetic diversity are vital to the evaluation of drug and vaccine efficacy, tracking of P. vivax outbreaks, and assessing geographical differentiation between parasite populations. METHODS: The genetic diversity of eight P. vivax populations (n=543) was investigated by using two microsatellites (MS), m1501 and m3502, chosen because of their seven and eight base-pair (bp) repeat lengths, respectively. These were compared with published data of the same loci from six other P. vivax populations. RESULTS: In total, 1,440 P. vivax samples from 14 countries on three continents were compared. There was highest heterozygosity within Asian populations, where expected heterozygosity (He) was 0.92-0.98, and alleles with a high repeat number were more common. Pairwise FST revealed significant differentiation between most P. vivax populations, with the highest divergence found between Asian and South American populations, yet the majority of the diversity (~89%) was found to exist within rather than between populations. CONCLUSIONS: The MS markers used were informative in both global and local P. vivax population comparisons and their seven and eight bp repeat length facilitated population comparison using data from independent studies. A complex spatial pattern of MS polymorphisms among global P. vivax populations was observed which has potential utility in future epidemiological studies of the P. vivax parasite.

PREVALENCE OF LYMPHATIC FILARIASIS, MALARIA AND SOIL TRANSMITTED HELMINTHIASIS IN A COMMUNITY OF BARDIYA DISTRICT, WESTERN NEPAL.

Lymphatic filariasis (LF), malaria and soil transmitted helminthiasis (STH) cause major health problems in Nepal, but in spite of this very few stud- ies have been carried out on these parasitic infections in Nepal. A cross sectional survey of all three categories of parasitic infections was carried out in Deuda- kala Village of Bardiya District, western Nepal. A total of 510 individuals aged 5 years and above were examined from finger prick blood for circulating filarial antigen (CFA), malaria antigen using a rapid diagnostic test (RDT), and malaria DNA using a PCR-based assay. In addition, 317 individuals were examined for soil-transmitted helminth (STH) eggs by the Kato-Katz technique. Prevalence of LF, malaria (antigen) and STH infection was 25.1%, 0.6% and 18.3%, respectively. PCR analysis did not detect any additional malaria cases. The prevalence of LF and STH infections differ significantly among different age groups and ethnic communities. The high prevalence of LF in the community studied indicates an immediate need for implementing a mass drug administration program for its control in this particular geographical area of Nepal.

Genetic diversity of plasmodium vivax merozoite surface protein-3alpha (Pvmsp-3alpha) gene in Jhapa District of Nepal.

In Nepal, Plasmodium vivax accounts for approximately 80-90% of the malaria cases, but limited studies have been conducted on the genetic diversity of this parasite population. This study was carried out to determine the genetic diversity of P. vivax population sampled from subjects living in an endemic area of Jhapa District by analyzing the polymorphic merozoite surface protein-3alpha (Pvmsp-3alpha) gene by using PCR-restriction fragment length polymorphism. Three distinct genotypes were obtained from 96 samples; type A: 40 (71%), type B: 7 (13%), and type C: 9 (16%) which could be categorized into 13 allelic patterns: A1-A9, B1, B2, C1 and C2. These results indicated a high genetic diversity within the studied P. vivax population. As the transmission rate of malaria is low in Nepal, the diversity is most likely due to migration of people between the malaria endemic regions, either within the country or between Nepal and India. Similar prevalence of the three genotypes of Pvmsp-3alpha between the two countries likely supports the latter explanation.

Prevalence of molecular markers of anti-malarial drug resistance in Plasmodium vivax and Plasmodium falciparum in two districts of Nepal.

BACKGROUND: Sulphadoxine-pyrimethamine (SP) and chloroquine (CQ) have been used in treatment of falciparum and vivax malaria in Nepal. Recently, resistance to both drugs have necessitated a change towards artemisinin combination therapy (ACT) against Plasmodium falciparum in highly endemic areas. However, SP is still used against P. falciparum infections in low endemic areas while CQ is used in suspected cases in areas with lack of diagnostic facilities. This study examines the prevalence of molecular markers of CQ and SP resistance in P. falciparum and Plasmodium vivax to determine if high levels of in vivo resistance are reflected at molecular level as well. METHODS: Finger prick blood samples (n=189) were collected from malaria positive patients from two high endemic districts and analysed for single nucleotide polymorphisms (SNPs) in the resistance related genes of P. falciparum and P. vivax for CQ (Pfcrt, Pfmdr1, Pvmdr1) and SP (Pfdhfr, Pfdhps, Pvdhfr), using various PCR-based methods. RESULTS AND DISCUSSION: Positive P. vivax and P. falciparum infections were identified by PCR in 92 and 41 samples respectively. However, some of these were negative in subsequent PCRs. Based on a few P. falciparum samples, the molecular level of CQ resistance in P. falciparum was high since nearly all parasites had the Pfcrt mutant haplotypes CVIET (55%) or SVMNT (42%), though frequency of the Pfmdr1 wild type haplotype was relatively low (35%). Molecular level of SP resistance in P. falciparum was found to be high. The most prevalent Pfdhfr haplotype was double mutant CNRNI (91%), while frequency of Pfdhps double mutant SGEAA and AGEAA were 38% and 33% respectively. Combined, the frequency of quadruple mutations (CNRNI-SGEAA/AGEAA) was 63%. Based on P. vivax samples, low CQ and SP resistance were most likely due to low prevalence of Pvmdr1 Y976F mutation (5%) and absence of triple/quadruple mutations in Pvdhfr. CONCLUSIONS: Based on the limited number of samples, prevalence of CQ and SP resistance at molecular levels in the population in the study area were determined as high in P. falciparum and low in P. vivax. Therefore, CQ could still be used in the treatment of P. vivax infections, but this remains to be tested in vivo while the change to ACT for P. falciparum seems justified.