Exposure to maternal feces in lactation influences piglet enteric microbiota, growth, and survival preweaning.

It is known that gilt progeny performance is reduced compared with sow progeny. Previous research suggests that the presence of maternal feces in early life improves the health and survival of offspring. Therefore, we aimed to determine whether contact with feces from multiparous (MP) sows would improve the growth and survival of piglets born and reared on primiparous (P1) sows and if so, whether these differences are associated with the gut microbiota. Four treatments were applied for 10 days: Donor (n = 29) piglets had limited access to maternal feces as, each morning, sow feces were removed and placed in the crate of a P1 sow (P1-FT; n = 30 piglets) and P1-Con (n = 29) and MP-Con (n = 33) piglets had access to their own mothers' feces. All piglets were weighed on days 1, 3, 10, and 18. Fecal samples were collected from a subset of sows (n = 10/treatment) 3 days post farrow and from two female piglets/litter on days 10 and 18 (n = 20/treatment) and subject to 16S rRNA amplicon analysis. Escherichia, Clostridium, Campylobacter, and Treponema were more abundant in MP sows, while P1 sows had a higher abundance of Lactobacillus and Prevotella. At 10 days, P1 progeny fecal microbiota differed, and growth and survival were reduced when compared with MP progeny. No treatment effect was observed for P1-FT piglets (P > 0.05). Donor piglets had a different fecal microbiota and improved weight and survival then all other treatments (P < 0.05). Overall, the removal of sow feces from the farrowing crate improved piglet microbiota development, growth, and survival.

Characterisation of Early Microbial Colonisers within the Spiral Colon of Pre- and Post-Natal Piglets.

Initial enteric microbial colonisation influences animal health and disease, hence an understanding of the first microbial colonisers within the piglet is important. The spiral colon of piglets that were stillborn (n = 20), born-alive (n = 10), and born alive and had sucked (n = 9) were collected from 28 sows to investigate whether initial microbial colonisation occurs pre- or post-partum and how it develops during the first 24 h post-partum. To examine this, DNA was extracted and 16S rRNA amplicon analysis was performed to allow analysis of microbial communities. The results indicate that microbial colonisation of the spiral colon had occurred in stillborn pigs, suggesting microbial exposure prior to birth. Alpha diversity metrics indicated that the number of taxa and community richness were higher in piglets that sucked (p < 0.001) and community evenness was lower in stillborns in comparison to born-alive (p < 0.001) but was not affected by colostrum consumption (p < 0.001). Additionally, when compared with stillborn piglets, the bacteria colonising the spiral colon during the first 24 h post-partum included the potentially pathogenic bacteria Escherichia coli, Clostridium perfringens and Clostridium celatum, and potentially beneficial bacteria Lactobacillus reutueri and Faecalibacterium prausnitzii. The relative presence of Archaea was high in stillborn piglets but decreased with post-natal environmental exposure. It is evident that stillborn piglets have bacteria present within their spiral colon, however further studies are needed in order to determine the time at which colonisation is initiated and the mechanisms determining how colonisation occurs. Additionally, as expected, the immediate post-natal environment largely influences the microorganisms colonising, while colostrum consumption further contributes to the microbial community enrichment.

A Single Faecal Microbiota Transplantation Altered the Microbiota of Weaned Pigs.

Weaning is a stressful time for piglets, often leading to weight loss and is associated with increased morbidity and mortality. A leading cause for these post-weaning problems is enteric dysbiosis and methods to improve piglet health at this crucial developmental stage are needed. This study aimed to determine whether an enteric dysbiosis caused by weaning could be corrected via a faecal microbiota transplantation (FMT) from healthy piglets from a previous wean. Two or four focal piglets per litter were assigned to one of two treatments; FMT two days post weaning (n = 21; FMT) or a control which received saline two days post weaning (n = 21; CON). FMT consisted of homogenised donor faeces administered orally at 3 mL/kg. Weaning occurred at 18 days of age and weights and faecal samples were collected on days 18, 20, 24 and 35. 16S rRNA amplicon analysis was used to assess the faecal microbiota of piglets. FMT increased Shannon's diversity post weaning (p < 0.001) and reduced the scratch score observed at 24 days of age (p < 0.001). The bacterial populations significantly differed in composition at each taxonomic level. In FMT pigs, significant increases in potentially pathogenic Escherichia coli were observed. However, increases in beneficial bacteria Lactobacillus mucosae and genera Fibrobacteres and Bacteroidetes were also observed in FMT treated animals. To our knowledge, this is the first study to observe a significant effect on piglet faecal microbiota following a single FMT administered post weaning. Therefore, FMT post weaning can potentially alleviate enteric dysbiosis.

Faecal Microbiota Analysis of Piglets During Lactation.

Antimicrobial use in animals and the potential development of antimicrobial resistance is a global concern. So, non-antimicrobial techniques for animal disease control are needed. This study aimed to determine whether neonatal ceftiofur (CF) treatment affects piglet faecal microbiomes and whether faecal microbiome transplantation (FMT) can correct it. Two focal piglets per sow were assigned to treatments as follows: cffresh (n = 6) received CF (3 mg/kg intramuscular) at 7 d and fresh FMT at 13 d; cffrozen (n = 7) received CF at 7 d and frozen FMT at 13 d; CF (n = 8) received CF at 7 d and no FMT; and no CF (n = 5) received no CF or FMT. DNA was extracted from faecal samples collected on days 7, 13, and 18 for 16S rRNA amplicon analysis. All faecal blends used for the FMT consisted of pooled donor pig faeces at 1:2 ratio with saline, delivered orally at 3 mL/kg. Alpha and beta diversity metrics increased with age (p < 0.05). However, no effect of antibiotic or FMT treatment was evident in 13 and 18 d old piglets (p > 0.05). Although no effect of treatment was observed, information regarding microbial membership during lactation was gained.

Measuring the Asian seabass (Lates calcarifer) neutrophil respiratory burst activity by the dihydrorhodamine-123 reduction flow cytometry assay in whole blood.

The neutrophil oxidative respiratory burst response is a key component of the innate immune system responsible for killing microbial pathogens. Since fish rely on the innate immune system for health, monitoring the respiratory burst activity may be an effective means of gauging fish health status. Here we report that the respiratory burst of Asian seabass neutrophils can be measured in whole blood by the dihydrorhodamine (DHR)-123 reduction assay and flow cytometry. Neutrophils responded to phorbol myristate acetate (PMA) in a concentration dependent manner with significant respiratory burst activity at 100-1000 nM. Other known neutrophil agonists, such as bacterial lipopolysaccharide, tumor necrosis factor, the tripeptide f-met-leu-phe and zymosan, did not induce a significant DHR reduction. Thus, the findings enable us to propose that the DHR-123 flow cytometry whole blood assay, incorporating PMA as a stimulator, would not only facilitate future studies into fish blood neutrophil research but provides a simple, rapid and reliable assay for gauging fish natural immunity status and health.

B-Cell Epitope Mapping Using a Library of Overlapping Synthetic Peptides in an Enzyme-Linked Immunosorbent Assay.

This chapter describes a strategy for mapping linear B-cell epitopes of proteins using synthetic biotinylated peptides in an ELISA.A set of overlapping peptides were designed based upon a known amino acid sequence of the target protein, VapA (Virulence-associated Protein A) of the bacterium Rhodococcus equi, an important pulmonary pathogen in foals. The peptides synthesized as biotinylated peptides were coated directly onto micro titer plates which had been pre-coated with NeutrAvidin™ and used to screen sera from foals confirmed to have R. equi disease. A linear B-cell epitope was identified which corresponded to a 20 mer sequence of the VapA protein.

An Adenoviral Vector Based Vaccine for Rhodococcus equi.

Rhodococcus equi is a respiratory pathogen which primarily infects foals and is endemic on farms around the world with 50% mortality and 80% morbidity in affected foals. Unless detected early and treated appropriately the disease can be fatal. Currently, there is no vaccine available to prevent this disease. For decades researchers have endeavoured to develop an effective vaccine to no avail. In this study a novel human adenoviral vector vaccine for R. equi was developed and tested in the mouse model. This vaccine generated a strong antibody and cytokine response and clearance of R. equi was demonstrated following challenge. These results show that this vaccine could potentially be developed further for use as a vaccine to prevent R. equi disease in foals.

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus Isolated from Australian Veterinarians.

This work investigated the molecular epidemiology and antimicrobial resistance of methicillin-resistant Staphylococcus aureus (MRSA) isolated from veterinarians in Australia in 2009. The collection (n = 44) was subjected to extensive molecular typing (MLST, spa, SCCmec, dru, PFGE, virulence and antimicrobial resistance genotyping) and antimicrobial resistance phenotyping by disk diffusion. MRSA was isolated from Australian veterinarians representing various occupational emphases. The isolate collection was dominated by MRSA strains belonging to clonal complex (CC) 8 and multilocus sequence type (ST) 22. CC8 MRSA (ST8-IV [2B], spa t064; and ST612-IV [2B], spa variable,) were strongly associated with equine practice veterinarians (OR = 17.5, 95% CI = 3.3-92.5, P < 0.001) and were often resistant to gentamicin and rifampicin. ST22-IV [2B], spa variable, were strongly associated with companion animal practice veterinarians (OR = 52.5, 95% CI = 5.2-532.7, P < 0.001) and were resistant to ciprofloxacin. A single pig practice veterinarian carried ST398-V [5C2], spa t1451. Equine practice and companion animal practice veterinarians frequently carried multiresistant-CC8 and ST22 MRSA, respectively, whereas only a single swine specialist carried MRSA ST398. The presence of these strains in veterinarians may be associated with specific antimicrobial administration practices in each animal species.

Impact of antibiotic use in the swine industry.

Antibiotic resistance in bacteria associated with pigs not only affects pig production but also has an impact on human health through the transfer of resistant organisms and associated genes via the food chain. This can compromise treatment of human infections. In the past most attention was paid to glycopeptide and streptogramin resistance in enterococci, fluoroquinolone resistance in campylobacter and multi-drug resistance in Escherichia coli and salmonella. While these are still important the focus has shifted to ESBL producing organisms selected by the use of ceftiofur and cefquinome in pigs. In addition Livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) suddenly emerged in 2007. We also need to consider multi-resistant strains of Streptococcus suis. Environmental contamination arising from piggery wastewater and spreading of manure slurry on pastures is also a growing problem.

The isolation and characterization of Campylobacter jejuni bacteriophages from free range and indoor poultry.

Six hundred and sixty one samples - primarily fresh chicken faeces - were processed to isolate wild type Campylobacter jejuni bacteriophages, via overlay agar methods using C. jejuni NCTC 12662. The aims of this study were to isolate and purify bacteriophages and then test for their ability to lyse field strains of C. jejuni in vitro. Of all samples processed, 130 were positive for bacteriophages. A distinct difference was observed between samples from different poultry enterprises. No bacteriophages could be isolated from indoor broilers. The majority of bacteriophages were isolated from free range poultry - both broilers and egg layers. Bacteriophages were purified and then selected for characterization based on their ability to produce clear lysis on plaque assay, as opposed to turbid plaques. Two hundred and forty one C. jejuni field isolates were tested for sensitivity to the bacteriophages. Lysis was graded subjectively and any minimal lysis was excluded. Using this system, 59.0% of the C. jejuni isolates showed significant sensitivity to at least one bacteriophage. The sensitivity to individual bacteriophages ranged from 10.0% to 32.5% of the C. jejuni isolates. Five bacteriophages were examined by electron microscopy and determined to belong to the Myoviridae family. The physical size, predicted genetic composition and genome size of the bacteriophages correlated well with other reported Campylobacter bacteriophages. The reasons for the observed difference between indoor broilers and free range poultry is unknown, but are postulated to be due to differences in the Campylobacter population in birds under different rearing conditions.

Antimicrobial and heavy metal resistance in commensal enterococci isolated from pigs.

Antibiotic resistance in animal isolates of enterococci is of public health concern because of the risk of transfer of antibiotic resistance isolates or resistance determinants to consumers via the food chain. In this study, phenotypic and genotypic resistance in 192 pig isolates of enterococci to ampicillin, avilamycin, avoparcin, bacitracin, flavophospholipol, gentamicin, narasin, tetracycline, tiamulin, tylosin, vancomycin, virginiamycin, copper and zinc were investigated by susceptibility test and molecular methods. Resistance rates varied between the species but all isolates were susceptible to ampicillin, avilamycin, avoparcin, gentamicin and narasin but resistant to tetracycline and tylosin and intermediately resistant to copper. Only Enterococcus gallinarum and Enterococcus casseliflavus were resistant to vancomycin and virginiamycin resistance was present in less than half the Enterococcus faecium isolates. Zinc resistance was largely confined to Enterococcus faecalis but bacitracin resistance was uncommon in E. faecalis in comparison with the other species. Tiamulin resistance was common in all species except E. casseliflavus. Resistance to flavophospholipol was detected in most E. faecium isolates and in a high proportion of E. gallinarum, E. casseliflavus and E. hirae/durans but was only found in one isolate of E. faecalis. No tetO, rplC, rplD, vanA, vanB, vatA and vatD genes were found. The presence of ermB, tetL, tetM, tcrB, aac6-aph2, tetK, tetS, vanC1, vanC2, lsaA, lsaB and vatE varied between the species and largely corresponded to the susceptibility phenotype. The findings show that resistance to antibiotics of high clinical significance for nosocomial Enterococcus infections is absent, whereas antimicrobial resistance was detected for some other antibiotics including bacitracin, flavophospholipol, tetracycline, tiamulin, tylosin and virginiamycin.

Whole-genome sequencing and gene mapping of a newly isolated lytic enterococcal bacteriophage EFRM31.

Bacteriophages contribute greatly to bacterial evolution. There has been limited investigation of enterococcal bacteriophages, and only two enterococcal bacteriophages have been sequenced completely. In this study, a novel enterococcal bacteriophage, EFRM31, was isolated from a piggery effluent sample and then characterized. The complete bacteriophage genome was determined by shotgun sequencing. EFRM31 belongs to the family Siphoviridae (order Caudovirales) and has a circular double-stranded DNA genome. The putative EFRM31 genome consists of 16945 nucleotides with a low GC content (34.5%) and does not contain CpG islands. The EFRM31 genome contains 82 putative open reading frames, including 17 with identities to genes required for the assembly of a head-tail bacteriophage and 6 hypothetical proteins of unknown function. In general, the sequencing results from EFRM31 revealed considerable similarity to another enterococcal bacteriophage, EFAP-1. This identity and the order of shared genes suggest a close relationship or a common ancestor for these two bacteriophages.

Novel Bacteriophages in Enterococcus spp.

Most of the bacteriophages (phages) currently reported in Enterococcus spp. belong to tailed families of bacteriophages Podoviridae, Siphoviridae, and Myoviridae. There is a little information on non-tailed bacteriophages isolated from enterococci. Samples of sewage and piggery effluents were tested on pig and chicken isolates of Enterococcus faecalis, E. faecium and E. gallinarum for lytic phages. In addition, isolates were exposed to mitomycin C to induce lysogenic phages. Bacteriophages that were detected were visualized by electron microscopy. Ten bacteriophages were of isometric shape with long flexible or non-flexible tails, while one had a long head with a long flexible tail; all contained double-stranded DNA molecules. Seven Polyhedral, filamentous, and pleomorphic-shaped phages containing DNA or RNA were also observed. The pleomorphic phages were droplet- or lemon-shaped in morphology. This study is the first report on polyhedral phages in Enterococcus spp. of animal origin and also the first report of filamentous and pleomorphic phages in enterococci.

Linear B-cell epitope mapping using enzyme-linked immunosorbent assay for libraries of overlapping synthetic peptides.

The aim of this chapter is to provide a strategy for mapping linear antibody epitopes of protein antigens in order to discover candidates for vaccines or diagnostic tests. A set of overlapping peptides was designed and synthesised based upon a known amino acid sequence of the target protein, virulence-associated protein A (VapA) of the bacterium Rhodococcus equi, an important pulmonary pathogen in foals.The peptides were biotinylated and used in an ELISA to screen immune sera from foals. These biotinylated peptides were coated directly onto micro titre plates that had been pre-coated with NeutrAvidin. A linear B-cell epitope was identified by a universal recognition of sera to the synthetic peptides which corresponds to a particular fragment of the VapA protein.

Chimeric vapA/groEL2 DNA vaccines enhance clearance of Rhodococcus equi in aerosol challenged C3H/He mice.

Rhodococcus equi remains a significant bacterial pathogen, causing severe pyogranulomatous pneumonia in foals aged 1-3 months. There is no effective vaccine currently available for the prevention of R. equi pneumonia. DNA vaccines are known to offer specific advantages over conventional vaccines. The aim of this study was to demonstrate efficacy of our recombinant DNA vaccine candidates, namely pcDNA3-Re1, pcDNA3-Re3 and pcDNA3-Re5 by combining a heat shock protein GroEL2 to a virulence-associated protein A (VapA) from R. equi to protect C3H/He mice against the R. equi infection. VapA was shown to be strongly recognised by sera from pneumonic foals. All vaccines elicited at least a doubling of the IgG2a/IgG1 ratio in comparison to the controls, indicating a bias to the Th1 response, which is postulated to be crucial for bacterial clearance and protective immunity against intracellular pathogens including R. equi. In addition, the immunised mice showed a significant reduction in R. equi in their lungs at 7 days after the aerosol challenge in comparison to PBS treated mice. However, examination of lung pathology 14 days after the challenge showed no gross differences in pathological changes between the unvaccinated and vaccinated animals. The lack of significant pathological changes suggests that the precise level of protection against R. equi pneumonia in the murine model of infection may not represent a true effectiveness of the potential vaccine candidates, indicating the mouse may not be the ideal non-equine model for vaccine studies and (or) the incomplete immunogenic antigen of vapA-based DNA vaccine constructs that mount an inadequate cell-mediated immune response against the R. equi infection.

Antimicrobial compounds from the Australian desert plant Eremophila neglecta.

A crude extract from the Australian desert plant Eremophila neglecta has recently been shown to possess antibacterial activity in a survey of candidate plants that may bear novel antimicrobial compounds. Bioassay-directed fractionation of the Et(2)O extract of E. neglecta using a broth microdilution assay led to the isolation of three new serrulatane-type diterpenoids, 2,19-diacetoxy-8-hydroxyserrulat-14-ene (2), 8,19-dihydroxyserrulat-14-ene (3), and 8-hydroxyserrulat-14-en-19-oic acid (4), and a known o-naphthoquinone commonly referred to as biflorin (5). The structures of 2-5 were determined using 1D and 2D NMR, FTIR, and high-resolution mass spectrometry. Compounds 3-5 showed antimicrobial activity against Gram-positive bacteria including Staphylococcus aureus, Streptococcus pyogenes, and S. pneumoniae. The minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) ranged from 6.5 to 101.6 microM and 12.7 to 202.9 microM, respectively. No activity was observed for these compounds against Gram-negative bacteria.

Antimicrobial compounds from Eremophila serrulata.

We report a search for antimicrobial compounds in the Australian plant Eremophila serrulata. Bioassay directed fractionation of a diethyl ether extract prepared from the leaves of E. serrulata led to the isolation of two compounds, an omicron-naphthoquinone, 9-methyl-3-(4-methyl-3-pentenyl)-2,3-dihydronaphtho[1,8-bc]pyran-7,8-dione (2), and a serrulatane diterpenoid, 20-acetoxy-8-hydroxyserrulat-14-en-19-oic acid (3). Two other known serrulatane-type diterpenoids, 8,20-dihydroxyserrulat-14-en-19-oic acid (4) and 8,20-diacetoxyserrulat-14-en-19-oic acid (5) were also isolated. None of these compounds had previously been tested for antimicrobial activity. Compounds 2-5 showed antimicrobial activity against Staphylococcus aureus (ATCC 29213) with minimum inhibitory concentrations (MICs) ranging from 15.6 to 250mug/mL. Compound 2 was the most active with an MIC of 15.6mug/mL and a minimum bactericidal concentration (MBC) of 125mug/mL. This compound also showed antimicrobial activity against other Gram-positive bacteria including Streptococcus pyogenes, and Streptococcus pneumoniae. No activity was observed for this compound against all Gram-negative bacteria tested.

Antibiotic and heavy metal resistance in motile aeromonads and pseudomonads from rainbow trout (Oncorhynchus mykiss) farms in Australia.

A total of 129 Pseudomonas spp. and 90 Aeromonas spp. were isolated from nine rainbow trout (Oncorhynchus mykiss) farms in Australia. All the isolates were tested for sensitivity to 15 antibiotics and the multiresistant strains were tested for sensitivity to seven heavy metals. Minimal inhibitory concentrations (MICs) were determined by the agar dilution method. In Pseudomonas spp., resistance to amoxicillin, cefalothin, ceftiofur, ticarcillin, chloramphenicol, florfenicol, streptomycin, nitrofurantoin and trimethoprim was widespread, whereas resistance to cefotaxime and oxolinic acid was less common and only single isolates were resistant to tetracycline and sulfamethoxazole; all isolates were sensitive to ciprofloxacin and gentamicin. In Aeromonas spp., resistance to amoxicillin and cefalothin was widespread, resistance to ticarcillin, tetracycline and streptomycin was common, whilst resistance to ceftiofur, florfenicol and sulfamethoxazole was less common. Single isolates were resistant to chloramphenicol, nitrofurantoin and trimethoprim, and all isolates were sensitive to cefotaxime, oxolinic acid, ciprofloxacin and gentamicin. Multiple resistance was also observed. Most isolates were tolerant to different concentrations of various heavy metals, as evidenced by their MICs ranging from 6.25 microg/mL to >3200 microg/mL. These results confirm our previous findings that bacteria resistant to antibiotics are present in fish and sediments from aquaculture in Australia. In addition, we have found resistance to heavy metals in fish and sediment isolates. Much of the antibiotic resistance detected is likely to be intrinsic, although resistance to oxytetracycline, streptomycin and sulfonamides suggests either contamination from run-off from farms or perhaps off-label use of antibiotics in a situation where no antibiotics are licensed for use in aquaculture.

Antimicrobial activity of some Australian plant species from the genus Eremophila.

Plant species of the genus Eremophila (Myoporaceae) are native to Australia and are known to produce a diverse range of unusual secondary compounds. The purpose of this research was to examine the antimicrobial activity of 72 Eremophila species most of which had not been the subject of any previous pharmacological testing. Organic extracts of Eremophila species were screened for antimicrobial activity against Gram-positive and Gram-negative bacteria and yeasts of medical importance. Extracts of a number of Eremophila species showed selective activity against Gram-positive bacteria with MICs for the most active species in the range of 16 to 62 microg/ml for Streptococcus species, and 62 to 250 microg/ml for standard strains of Staphylococcus aureus. Extracts with the greatest activity against standard strains were tested against 68 clinical isolates of multi-resistant methicillin-resistant S. aureus (mMRSA). The majority of the clinical isolates were susceptible to concentrations below 62.5 microg/ml for the extracts of E. drummondii, E. linearis, E. serrulata, E. acrida, E. neglecta, E. virens and a new undescribed species affiliated with E. prolata. The extract of E. virens inhibited growth of all 68 clinical mMRSA isolates at the minimum tested concentration of 31 microg/ml. This study has shown for the first time that a number of different Eremophila species manifest selective antibacterial activity against Gram-positive organisms which are important causes of human disease. It shows that there are several Eremophila species possessing interesting antibacterial activity besides those that have published traditional use. These may yield novel antibacterial compounds with potential to be used in biomedical applications.