Mikael Lenz Strube

Natural Pig Plasma Immunoglobulins Have Anti-Bacterial Effects: Potential for Use as Feed Supplement for Treatment of Intestinal Infections in Pigs.

There is an increasing demand for non-antibiotics solutions to control infectious disease in intensive pig production. Here, one such alternative, namely pig antibodies purified from slaughterhouse blood was investigated in order to elucidate its potential usability to control post-weaning diarrhoea (PWD), which is one of the top indications for antibiotics usage in the pig production. A very cost-efficient and rapid one-step expanded bed adsorption (EBA) chromatography procedure was used to purify pig immunoglobulin G from slaughterhouse pig plasma (more than 100 litres), resulting in >85% pure pig IgG (ppIgG). The ppIgG thus comprised natural pig immunoglobulins and was subsequently shown to contain activity towards four pig-relevant bacterial strains (three different types of Escherichia coli and one type of Salmonella enterica) but not towards a fish pathogen (Yersinia ruckeri), and was demonstrated to inhibit the binding of the four pig relevant bacteria to a pig intestinal cell line (IPEC-J2). Finally it was demonstrated in an in vivo weaning piglet model for intestinal colonization with an E. coli F4+ challenge strain that ppIgG given in the feed significantly reduced shedding of the challenge strain, reduced the proportion of the bacterial family Enterobacteriaceae, increased the proportion of families Enterococcoceae and Streptococcaceae and generally increased ileal microbiota diversity. Conclusively, our data support the idea that natural IgG directly purified from pig plasma and given as a feed supplement can be used in modern swine production as an efficient and cost-effective means for reducing both occurrence of PWD and antibiotics usage and with a potential for the prevention and treatment of other intestinal infectious diseases even if the causative agent might not be known.

Characterization of the bacterial gut microbiota of piglets suffering from new neonatal porcine diarrhoea

BackgroundIn recent years, new neonatal porcine diarrhoea (NNPD) of unknown aetiology has emerged in Denmark. NNPD affects piglets during the first week of life and results in impaired welfare, decreased weight gain, and in the worst-case scenario death. Commonly used preventative interventions such as vaccination or treatment with antibiotics, have a limited effect on NNPD. Previous studies have investigated the clinical manifestations, histopathology, and to some extent, microbiological findings; however, these studies were either inconclusive or suggested that Enterococci, possibly in interaction with Escherichia coli, contribute to the aetiology of NNPD. This study examined ileal and colonic luminal contents of 50 control piglets and 52 NNPD piglets by means of the qPCR-based Gut Microbiotassay and 16 samples by 454 sequencing to study the composition of the bacterial gut microbiota in relation to NNPD.ResultsNNPD was associated with a diminished quantity of bacteria from the phyla Actinobacteria and Firmicutes while genus Enterococcus was more than 24 times more abundant in diarrhoeic piglets. The number of bacteria from the phylum Fusobacteria was also doubled in piglets suffering from diarrhoea. With increasing age, the gut microbiota of NNPD affected piglet and control piglets became more diverse. Independent of diarrhoeic status, piglets from first parity sows (gilts) possessed significantly more bacteria from family Enterobacteriaceae and species E. coli, and fewer bacteria from phylum Firmicutes. Piglets born to gilts had 25 times higher odds of having NNPD compared with piglets born to multiparous sows. Finally, the co-occurrence of genus Enterococcus and species E. coli contributed to the risk of having NNPD.ConclusionThe results of this study support previous findings that points towards genus Enterococcus and species E. coli to be involved in the pathogenesis of NNPD. Moreover, the results indicate that NNPD is associated with a disturbed bacterial composition and larger variation between the diarrhoeic piglets.

Potential bacterial core species associated with digital dermatitis in cattle herds identified by molecular profiling of interdigital skin samples

Although treponemes are consistently identified in tissue from bovine digital dermatitis (DD) lesions, the definitive etiology of this debilitating polymicrobial disease is still unresolved. To study the microbiomes of 27 DD-infected and 10 healthy interdigital skin samples, we used a combination of different molecular methods. Deep sequencing of the 16S rRNA gene variable regions V1-V2 showed that Treponema, Mycoplasma, Fusobacterium and Porphyromonas were the genera best differentiating the DD samples from the controls. Additional deep sequencing analysis of the most abundant genus, Treponema, targeting another variable region of the 16S rRNA gene, V3-V4, identified 15 different phylotypes, among which Treponema phagedenis-like and Treponema refringens-like species were the most abundant. Although the presence of Treponema spp., Fusobacterium necrophorum and Porphyromonas levii was confirmed by fluorescence in situ hybridization (FISH), the results for Mycoplasma spp. were inconclusive. Extensive treponemal epidermal infiltration, constituting more than 90% of the total bacterial population, was observed in 24 of the 27 DD samples. F. necrophorum and P. levii were superficially located in the epidermal lesions and were present in only a subset of samples. RT-qPCR analysis showed that treponemes were also actively expressing a panel of virulence factors at the site of infection. Our results further support the hypothesis that species belonging to the genus Treponema are major pathogens of DD and also provide sufficient clues to motivate additional research into the role of M. fermentans, F. necrophorum and P. levii in the etiology of DD.

In Situ Prebiotics for Weaning Piglets: In Vitro Production and Fermentation of Potato Galacto-Rhamnogalacturonan

ABSTRACT Postweaning diarrhea (PWD) in pigs is a leading cause of economic loss in pork production worldwide. The current practice of using antibiotics and zinc to treat PWD is unsustainable due to the potential of antibiotic resistance and ecological disturbance, and novel methods are required. In this study, an in vitro model was used to test the possibility of producing prebiotic fiber in situ in the gastrointestinal (GI) tract of the piglet and the prebiotic activity of the resulting fiber in the terminal ileum. Soluble fiber was successfully produced from potato pulp, an industrial waste product, with the minimal enzyme dose in a simulated upper GI tract model extracting 26.9% of the initial dry matter. The fiber was rich in galactose and galacturonic acid and was fermented at 2.5, 5, or 10 g/liter in a glucose-free medium inoculated with the gut contents of piglet terminal ileum. Fermentations of 5 g/liter inulin or 5 g/liter of a purified potato fiber were used as controls. The fibers showed high fermentability, evident by a dose-dependent drop in pH and an increase in the organic acid content, with lactate in particular being increased. Deep sequencing showed a significant increase in the numbers of Lactobacillus and Veillonella organisms and an insignificant increase in the numbers of Clostridium organisms as well as a decrease in the numbers of Streptococcus organisms. Multivariate analysis showed clustering of the treatment groups, with the group treated with purified potato fiber being clearly separated from the other groups, as the microbiota composition was 60% Lactobacillus and almost free of Clostridium. For animal studies, a dosage corresponding to the 5-g/liter treatment is suggested.

Sequence-Based Genotyping of Expressed Swine Leukocyte Antigen Class I Alleles by Next-Generation Sequencing Reveal Novel Swine Leukocyte Antigen Class I Haplotypes and Alleles in Belgian, Danish, and Kenyan Fattening Pigs and Göttingen Minipigs

The need for typing of the swine leukocyte antigen (SLA) is increasing with the expanded use of pigs as models for human diseases and organ-transplantation experiments, their use in infection studies, and for design of veterinary vaccines. Knowledge of SLA sequences is furthermore a prerequisite for the prediction of epitope binding in pigs. The low number of known SLA class I alleles and the limited knowledge of their prevalence in different pig breeds emphasizes the need for efficient SLA typing methods. This study utilizes an SLA class I-typing method based on next-generation sequencing of barcoded PCR amplicons. The amplicons were generated with universal primers and predicted to resolve 68–88% of all known SLA class I alleles dependent on amplicon size. We analyzed the SLA profiles of 72 pigs from four different pig populations; Göttingen minipigs and Belgian, Kenyan, and Danish fattening pigs. We identified 67 alleles, nine previously described haplotypes and 15 novel haplotypes. The highest variation in SLA class I profiles was observed in the Danish pigs and the lowest among the Göttingen minipig population, which also have the highest percentage of homozygote individuals. Highlighting the fact that there are still numerous unknown SLA class I alleles to be discovered, a total of 12 novel SLA class I alleles were identified. Overall, we present new information about known and novel alleles and haplotypes and their prevalence in the tested pig populations.

Atlantic salmon (Salmo salar) protein hydrolysate in diets for weaning piglets ─ effect on growth performance, intestinal morphometry and microbiota composition

Abstract Salmon protein hydrolysates (SPH) from two different rest raw materials were evaluated in diets for weaning piglets. Four experimental diets were included in the study: a diet based on plant protein with soy protein as the main protein source (Diet PP), a diet based on fishmeal in exchange for soy protein (Diet FM) and two diets in which different SPH replaced fishmeal in the FM diet. The experimental diets were fed to piglets from the day of weaning until 32 d postweaning. In addition to the record of performance data, an intestinal sampling for mucosal morphometry and microbiota 16S rRNA gene sequencing were performed at day 11 on a subset of the animals. The duodenal villi absorption area was significantly larger in piglets receiving Diets SPH compared with Diet PP (p < 0.02). A significant positive correlation between duodenal villi height and average daily gain during the first 11 d postweaning was detected. Only small differences in intestinal microbiota community and no differences in growth performance were detected between the experimental diets. To conclude, SPH seem to be an interesting novel protein source in weanling piglets.

Microbiota Analysis of an Environmental Slurry and Its Potential Role as a Reservoir of Bovine Digital Dermatitis Pathogens

ABSTRACT At present, very little information exists regarding what role the environmental slurry may play as an infection reservoir and/or route of transmission for bovine digital dermatitis (DD), a disease which is a global problem in dairy herds. To investigate whether DD-related bacteria belong to the indigenous microbiota of the dairy herd environment, we used deep amplicon sequencing of the 16S rRNA gene in 135 slurry samples collected from different sites in 22 dairy farms, with and without DD-infected cows. Both the general bacterial populations and digital dermatitis-associated Treponema were targeted in this study. The results revealed significant differences in the bacterial communities between the herds, with only 12 bacterial taxa shared across at least 80% of all the individual samples. These differences in the herd microbiota appeared to reflect mainly between-herd variation. Not surprisingly, the slurry was dominated by ubiquitous gastrointestinal bacteria, such as Ruminococcaceae and Lachnospiraceae. Despite the low relative abundance of spirochetes, which ranged from 0 to 0.6%, we were able to detect small amounts of bacterial DNA from DD-associated treponemes in the slurry. However, the DD-associated Treponema spp. were detected only in samples from herds with reported DD problems. These data indicate that treponemes involved in the pathogenesis of DD are not part of the normal environmental microflora in dairy herds without clinical DD and, consequently, that slurry is not a primary reservoir of infection. IMPORTANCE Bovine digital dermatitis (DD), a dermal disease which causes lameness in dairy cattle, is a serious problem worldwide. To control this disease, the infection reservoirs and transmission routes of DD pathogens need to be clarified. The dairy herd slurry may be a pathogen reservoir of DD-associated bacteria. The rationale for the present study was, therefore, to examine whether DD-associated bacteria are always present in slurry or if they are found only in DD-afflicted herds. The results strongly indicated that DD Treponema spp. are not part of the indigenous slurry and, therefore, do not comprise an infection reservoir in healthy herds. This study applied next-generation sequencing technology to decipher the microbial compositions of environmental slurry of dairy herds with and without digital dermatitis.

A detailed investigation of the porcine skin and nose microbiome using universal and Staphylococcus specific primers

MRSA is an increasing problem in humans as well as livestock. The bacterial co-colonization of the skin in MRSA carriers has been poorly investigated and moreover, there have been no methods for high resolution investigations of the Staphylococcus genus apart from tediously culturing or doing multiple PCRs. On 120 samples from pig ear, skin and nose, we generated amplicons from the V1-V2 region of the 16S rRNA gene to gather an overview of the genus-level microbiome, along with using MRSA specific plates to count MRSA. In parallel with this, amplicons of the tuf gene were generated, targeting only a region of the tuf gene found only in the Staphylococcus genus. Using these methods, we determined a core microbiota across the healthy pig and determined the Staphylococcus genus to be dominated by S. equorum. Moreover, we found Streptococcus to be inversely associated with Staphylococcus and MRSA, suggesting a role for this genus in combating MRSA. In this work, we have thoroughly investigated the skin and nose microbiome of the pig and developed a high throughput method for profiling the Staphylococcus genus which we believe will be useful for further investigations.

Pathology and bacteria related to digital dermatitis in dairy cattle in all year round grazing system in Brazil

Digital dermatitis (DD) is one of the main causes of lameness in dairy cattle worldwide, and it is frequently reported in high-yielding, free stall dairy herds from regions with a temperate climate. However, DD is also observed with high prevalence in grazing cattle with a low milk yield in tropical regions. To clarify whether these differences have an impact on the etiology of the disease, we studied DD lesions from all year round grazing cattle of mixed breed in Brazil using high-throughput 16S rRNA gene sequencing and fluorescent in situ hybridization. The study included samples from 66 skin lesions and 5 healthy skins collected from five farms. Both techniques showed Treponema spp. to be the most abundant bacteria, present in all but one of the samples with minimal epidermal alterations. We identified eleven different Treponema strains belonging to the six major phylotypes of Treponema which have all previously been identified in DD lesions. Furthermore, we identify Dichelobacter nodosus in DD lesions by gene sequencing and also by fluorescent in situ hybridization in almost half of biopsy specimens in areas with mild epithelial damage and together with Treponema. The present data support the hypothesis that Treponema constitutes the main pathogen responsible for DD, independent of the environment and region where cows are kept, and it further suggests D. nodosus as another potentially important pathogen.

Comparing low volume saphenous-obturator block with placebo and femoral-obturator block for anterior cruciate ligament reconstruction: a randomized controlled trial

BACKGROUND Anterior cruciate ligament reconstruction (ACL-RC) is often associated with moderate to severe postoperative pain even with a multimodal analgesic regimen. We aimed to compare the analgesic efficacy of low volume saphenous-obturator block with placebo and femoral-obturator block in patients undergoing ACL-RC. METHODS In a randomized controlled trial eighty-two patients undergoing ACL-RC with hamstring autograft were allocated to either low volume saphenous-obturator block, placebo block or femoral-obturator block. Ropivacaine 0.75% was used for active blocks and saline for placebo. Primary outcome was pain-scores at rest quantified as area-under-the-curve 0-6 hr postoperatively. Secondary outcomes were postoperative opioid consumption and pain localization in the knee. RESULTS No statistical difference existed between groups in area-under-the-curve 6 hr pain-scores. However, pain-scores were significantly lower in the two ropivacaine groups compared to placebo at emergence t=0 (P<0.018), at t=5 (P<0.042) and at t=6 hours (P<0.002) postoperatively. Furthermore, ropivacaine blocks exhibited significantly reduced total opioid consumption (15.81 and 18.44 mg) postoperatively compared with placebo (26.38 mg) (P<0.018). Patients receiving ropivacaine blocks localized pain in the posterolateral knee, whereas placebo block patients reported anteromedial and central pain. Other secondary outcomes were similar between groups. CONCLUSIONS Low volume saphenous-obturator block is significantly more effective than placebo in reducing both early and late pain-scores as well as postoperative opioid consumption in patients undergoing ACL-RC. No statistical difference existed when comparing low volume saphenous-obturator block to femoral-obturator block regarding early and late pain-scores and postoperative opioid consumption.