Genetic variability and antimicrobial resistance of Ureaplasma parvum in response to maternal erythromycin treatment : a study in pregnant sheep

Background: Ureaplasmas are the most frequently isolated microorganisms from the amniotic fluid (AF) of pregnant women and can cause chronic infections that are difficult to eradicate with standard macrolide treatment. We tested the effects of erythromycin treatment on phenotypic and genotypic markers of ureaplasmal antimicrobial resistance in sheep. Method: At 50 days of gestation (d, term=145d) 12 pregnant ewes received intra-amniotic injections of U. parvum serovar 3 (erythromycin-sensitive, 2x104 colony-forming-units). At 100d ewes received: erythromycin treatment (500 mg, q3h for 4 days, IM, n=6) or no treatment (n=6). Fetuses were delivered surgically (125d) and AF and chorioamnion were collected for: culture, minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC) testing; 23S rRNA sequencing; and detection of macrolide-lincosamide-streptogramin resistance (MLSr) genes. Results: MICs of erythromycin, azithromycin and roxithromycin against AF isolates were low (range = 0.06 mg/L to 1.0 mg/L); however, chorioamnion isolates demonstrated increased resistance to roxithromycin (0.13 – 5.33 mg/L). 62.5% of chorioamnion ureaplasmas formed biofilms in vitro and mutations (125 nucleotides, 29.6%) were found in the 23S rRNA gene (domain V) of chorioamnion (but not AF) ureaplasmas. MLSr genes (ermB, msrC and msrD) were detected in 100% of chorioamnion isolates and only msrD was detected in AF isolates (40%). Conclusions: 23S rRNA mutations and MLSr genes occurred independently of erythromycin treatment, suggesting that the anatomical site of infection and microenvironment may exert selective pressures on ureaplasmas that cause genetic changes and alter antimicrobial sensitivity profiles. These results have serious implications for treatment of in utero infections.

A new model to study healing of a complex femur fracture with concurrent soft tissue injury in sheep

High energy bone fractures resulting from impact trauma are often accompanied by subcutaneous soft tissue injuries, even if the skin remains intact. There is evidence that such closed soft tissue injuries affect the healing of bone fractures, and vice versa. Despite this knowledge, most impact trauma studies in animals have focussed on bone fractures or soft tissue trauma in isolation. However, given the simultaneous impact on both tissues a better understanding of the interaction between these two injuries is necessary to optimise clinical treatment. The aim of this study was therefore to develop a new experimental model and characterise, for the first time, the healing of a complex fracture with concurrent closed soft tissue trauma in sheep. A pendulum impact device was designed to deliver a defined and standardised impact to the distal thigh of sheep, causing a reproducible contusion injury to the subcutaneous soft tissues. In a subsequent procedure, a reproducible femoral butterfly fracture (AO C3-type) was created at the sheep’s femur, which was initially stabilised for 5 days by an external fixator construct to allow for soft tissue swelling to recede, and ultimately in a bridging construct using locking plates. The combined injuries were applied to twelve sheep and the healing observed for four or eight weeks (six animals per group) until sacrifice. The pendulum impact led to a moderate to severe circumferential soft tissue injury with significant bruising, haematomas and partial muscle disruptions. Posttraumatic measurements showed elevated intra-compartmental pressure and circulatory tissue breakdown markers, with recovery to normal, pre-injury values within four days. Clinically, no neurovascular deficiencies were observed. Bi-weekly radiological analysis of the healing fractures showed progressive callus healing over time, with the average number of callus bridges increasing from 0.4 at two weeks to 4.2 at eight weeks. Biomechanical testing after sacrifice showed increasing torsional stiffness between four and eight weeks healing time from 10% to 100%, and increasing ultimate torsional strength from 10% to 64% (relative to the contralateral control limb). Our results demonstrate the robust healing of a complex femur fracture in the presence of a severe soft tissue contusion injury in sheep and demonstrate the establishment of a clinically relevant experimental model, for research aimed at improving the treatment of bone fractures accompanied by closed soft tissue injuries.

Autologous vs. allogenic mesenchymal progenitor cells for the reconstruction of critical-sized segmental tibial bone defects in aged sheep

Mesenchymal progenitor cells (MPCs) represent an attractive cell population for bone tissue engineering. Their special immunological characteristics suggest that MPCs may be used in an allogenic application. The objective of this study was to compare the regenerative potential of autologous vs. allogenic MPCs in an ovine critical-sized segmental defect model. Ovine MPCs were isolated from bone marrow aspirates, expanded and cultured with osteogenic media for two weeks before implantation. Autologous and allogenic transplantation was performed by using the cell-seeded scaffolds, unloaded scaffolds and the application of autologous bone grafts served as control groups (n=6). Bone healing was assessed twelve weeks after surgery by radiology, micro computed tomography, biomechanical testing and histology. Radiology, biomechanical testing and histology revealed no significant difference in bone formation between the autologous and allogenic group. Both cell groups showed more bone formation than the scaffold alone, whereas the biomechanical data showed no significant differences between the cell-groups and the unloaded scaffolds. The results of the study suggest that scaffold based bone tissue engineering using allogenic cells offers the potential for an off the shelf product. Therefore, the results of this study serve as an important baseline for the translation of the assessed concepts into clinical application.

Ureaplasma parvum undergoes selection in utero resulting in genetically diverse isolates colonising the chorioamnion of fetal sheep

Ureaplasmas are the microorganisms most frequently isolated from the amniotic fluid of pregnant women and can cause chronic intrauterine infections. These tiny bacteria are thought to undergo rapid evolution and exhibit a hypermutatable phenotype; however, little is known about how ureaplasmas respond to selective pressures in utero. Using an ovine model of chronic intra-amniotic infection, we investigated if exposure of ureaplasmas to sub-inhibitory concentrations of erythromycin could induce phenotypic or genetic indicators of macrolide resistance. At 55 days gestation, 12 pregnant ewes received an intra-amniotic injection of a non-clonal, clinical U. parvum strain, followed by: (i) erythromycin treatment (IM, 30 mg/kg/day, n=6); or (ii) saline (IM, n=6) at 100 days gestation. Fetuses were then delivered surgically at 125 days gestation. Despite injecting the same inoculum into all ewes, significant differences between amniotic fluid and chorioamnion ureaplasmas were detected following chronic intra-amniotic infection. Numerous polymorphisms were observed in domain V of the 23S rRNA gene of ureaplasmas isolated from the chorioamnion (but not the amniotic fluid), resulting in a mosaic-like sequence. Chorioamnion isolates also harboured the macrolide resistance genes erm(B) and msr(D) and were associated with variable roxithromycin minimum inhibitory concentrations. Remarkably, this variability occurred independently of exposure of ureaplasmas to erythromycin, suggesting that low-level erythromycin exposure does not induce ureaplasmal macrolide resistance in utero. Rather, the significant differences observed between amniotic fluid and chorioamnion ureaplasmas suggest that different anatomical sites may select for ureaplasma sub-types within non-clonal, clinical strains. This may have implications for the treatment of intrauterine ureaplasma infections.

Multilocus sequence analysis provides insights into molecular epidemiology of Chlamydia pecorum infections in Australian sheep, cattle and koalas

Chlamydia pecorum is a significant pathogen of domestic livestock and wildlife. We have developed a C. pecorum-specific multilocus sequence analysis (MLSA) scheme to examine the genetic diversity of and relationships between Australian sheep, cattle, and koala isolates. An MLSA of seven concatenated housekeeping gene fragments was performed using 35 isolates, including 18 livestock isolates (11 Australian sheep, one Australian cow, and six U.S. livestock isolates) and 17 Australian koala isolates. Phylogenetic analyses showed that the koala isolates formed a distinct clade, with limited clustering with C. pecorum isolates from Australian sheep. We identified 11 MLSA sequence types (STs) among Australian C. pecorum isolates, 10 of them novel, with koala and sheep sharing at least one identical ST (designated ST2013Aa). ST23, previously identified in global C. pecorum livestock isolates, was observed here in a subset of Australian bovine and sheep isolates. Most notably, ST23 was found in association with multiple disease states and hosts, providing insights into the transmission of this pathogen between livestock hosts. The complexity of the epidemiology of this disease was further highlighted by the observation that at least two examples of sheep were infected with different C. pecorum STs in the eyes and gastrointestinal tract. We have demonstrated the feasibility of our MLSA scheme for understanding the host relationship that exists between Australian C. pecorum strains and provide the first molecular epidemiological data on infections in Australian livestock hosts.

Polycaprolactone scaffold and reduced rhBMP-7 dose for the regeneration of critical-sized defects in sheep tibiae

The transplantation of autologous bone graft as a treatment for large bone defects has the limitation of harvesting co-morbidity and limited availability. This drives the orthopaedic research community to develop bone graft substitutes. Routinely, supra-physiological doses of bone morphogenetic proteins (BMPs) are applied perpetuating concerns over undesired side effects and cost of BMPs. We therefore aimed to design a composite scaffold that allows maintenance of protein bioactivity and enhances growth factor retention at the implantation site. Critical-sized defects in sheep tibiae were treated with the autograft and with two dosages of rhBMP-7, 3.5 mg and 1.75 mg, embedded in a slowly degradable medical grade poly(ε-caprolactone) (PCL) scaffold with β-tricalcium phosphate microparticles (mPCL-TCP). Specimens were characterised by biomechanical testing, microcomputed tomography and histology. Bridging was observed within 3 months for the autograft and both rhBMP-7 treatments. No significant difference was observed between the low and high rhBMP-7 dosages or between any of the rhBMP-7 groups and autograft implantation. Scaffolds alone did not induce comparable levels of bone formation compared to the autograft and rhBMP-7 groups. In summary, the mPCL-TCP scaffold with the lower rhBMP-7 dose led to equivalent results to autograft transplantation or the high BMP dosage. Our data suggest a promising clinical future for BMP application in scaffold-based bone tissue engineering, lowering and optimising the amount of required BMP.

Comparative genomics of koala, cattle and sheep strains of Chlamydia pecorum

Background Chlamydia pecorum is an important pathogen of domesticated livestock including sheep, cattle and pigs. This pathogen is also a key factor in the decline of the koala in Australia. We sequenced the genomes of three koala C. pecorum strains, isolated from the urogenital tracts and conjunctiva of diseased koalas. The genome of the C. pecorum VR629 (IPA) strain, isolated from a sheep with polyarthritis, was also sequenced. Results Comparisons of the draft C. pecorum genomes against the complete genomes of livestock C. pecorum isolates revealed that these strains have a conserved gene content and order, sharing a nucleotide sequence similarity > 98%. Single nucleotide polymorphisms (SNPs) appear to be key factors in understanding the adaptive process. Two regions of the chromosome were found to be accumulating a large number of SNPs within the koala strains. These regions include the Chlamydia plasticity zone, which contains two cytotoxin genes (toxA and toxB), and a 77 kbp region that codes for putative type III effector proteins. In one koala strain (MC/MarsBar), the toxB gene was truncated by a premature stop codon but is full-length in IPTaLE and DBDeUG. Another five pseudogenes were also identified, two unique to the urogenital strains C. pecorum MC/MarsBar and C. pecorum DBDeUG, respectively, while three were unique to the koala C. pecorum conjunctival isolate IPTaLE. An examination of the distribution of these pseudogenes in C. pecorum strains from a variety of koala populations, alongside a number of sheep and cattle C. pecorum positive samples from Australian livestock, confirmed the presence of four predicted pseudogenes in koala C. pecorum clinical samples. Consistent with our genomics analyses, none of these pseudogenes were observed in the livestock C. pecorum samples examined. Interestingly, three SNPs resulting in pseudogenes identified in the IPTaLE isolate were not found in any other C. pecorum strain analysed, raising questions over the origin of these point mutations. Conclusions The genomic data revealed that variation between C. pecorum strains were mainly due to the accumulation of SNPs, some of which cause gene inactivation. The identification of these genetic differences will provide the basis for further studies to understand the biology and evolution of this important animal pathogen. Keywords: Chlamydia pecorum; Single nucleotide polymorphism; Pseudogene; Cytotoxin

Mimicry of 21. 5 KDa myelin basic protein peptide by a Maedi Visna virus polymerase peptide does not contribute to the pathogenesis of encephalitis in sheep

Epitope mimicry is the theory that an infectious agent such as a virus causes pathological effects via mimicry of host proteins and thus elicits a cross-reactive immune response to host tissues. Weise and Carnegie (1988) found a region of sequence similarity between the pol gene of the Maedi Visna virus (MVV), which induces demyelinating encephalitis in sheep, and myelin basic protein (MBP), which is known to induce experimental allergic encephalitis (EAE) in laboratory animals. In this study, cross-reactions between sera raised in sheep against synthetic peptides of MVV (TGKIPWILLPGR) and 21.5 kDa MBP (SGKVPWLKRPGR) were demonstrated using enzyme-linked immunosorbant assay (ELISA) and thin layer chromatography (TLC) immunoprobing. The antibody responses of MVV-infected sheep were investigated using ELISA against the peptides, and MBP protein, immunoprobing of the peptides on TPC plates and Western blotting against MBP. Slight significant reactions to the 21.5 kDa MBP peptide (P < 0.001) and to a lesser extent sheep MBP (P < 0.004) were detected in ELISA. The MBP peptide evoked stronger responses from more sera than the MVV peptide on immunoprobed TLC plates. On the Western blots, eight of the 23 sheep with Visna had serum reactivity to MBP. This slight reaction to MBP in MVV-infected sheep is of interest because of the immune responses to MBP evident in multiple sclerosis and EAE, but its relevance in Visna is limited since no correlation with disease severity was observed. The cell-mediated immune responses of MVV-infected sheep against similar peptides was assessed. The peptides did not stimulate proliferation of peripheral blood lymphocytes of MVV-infected sheep. Since the MVV peptide was not recognised by antibodies or T lymphocytes from MVV-infected and encephalic sheep, it was concluded that epitope mimicry of this 21.5 kDa MBP peptide by the similar MVV pol peptide was not contributing to the immunopathogenesis of Visna. The slight antibody response to MBP and the MBP peptide can be attributed to by-stander effects of the immunopathology of MVV-induced encephalitis.

A polymerase chain reaction-based method for isolating clones from a complimentary DNA library in sheep

The sheep (Ovis aries) is favored by many musculoskeletal tissue engineering groups as a large animal model because of its docile temperament and ease of husbandry. The size and weight of sheep are comparable to humans, which allows for the use of implants and fixation devices used in human clinical practice. The construction of a complimentary DNA (cDNA) library can capture the expression of genes in both a tissue- and time-specific manner. cDNA libraries have been a consistent source of gene discovery ever since the technology became commonplace more than three decades ago. Here, we describe the construction of a cDNA library using cells derived from sheep bones based on the pBluescript cDNA kit. Thirty clones were picked at random and sequenced. This led to the identification of a novel gene, C12orf29, which our initial experiments indicate is involved in skeletal biology. We also describe a polymerase chain reaction-based cDNA clone isolation method that allows the isolation of genes of interest from a cDNA library pool. The techniques outlined here can be applied in-house by smaller tissue engineering groups to generate tools for biomolecular research for large preclinical animal studies and highlights the power of standard cDNA library protocols to uncover novel genes.

Proteogenomics of selective susceptibility to endotoxin using circulating acute phase biomarkers and bioassay development in sheep: a review

Scientists have injected endotoxin into animals to investigate and understand various pathologies and novel therapies for several decades. Recent observations have shown that there is selective susceptibility to Escherichia coli lipopolysaccharide (LPS) endotoxin in sheep, despite having similar breed characteristics. The reason behind this difference is unknown, and has prompted studies aiming to explain the variation by proteogenomic characterisation of circulating acute phase biomarkers. It is hypothesised that genetic trait, biochemical, immunological and inflammation marker patterns contribute in defining and predicting mammalian response to LPS. This review discusses the effects of endotoxin and host responses, genetic basis of innate defences, activation of the acute phase response (APR) following experimental LPS challenge, and the current approaches employed in detecting novel biomarkers including acute phase proteins (APP) and micro-ribonucleic acids (miRNAs) in serum or plasma. miRNAs are novel targets for elucidating molecular mechanisms of disease because of their differential expression during pathological, and in healthy states. Changes in miRNA profiles during a disease challenge may be reflected in plasma. Studies show that gel-based two-dimensional electrophoresis (2-DE) coupled with either matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) or liquid chromatography-mass spectrometry (LC-MS/MS) are currently the most used methods for proteome characterisation. Further evidence suggests that proteomic investigations are preferentially shifting from 2-DE to non-gel based LC-MS/MS coupled with data extraction by sequential window acquisition of all theoretical fragment-ion spectra (SWATH) approaches that are able to identify a wider range of proteins. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and most recently proteomic methods have been used to quantify low abundance proteins such as cytokines. qRT-PCR and next generation sequencing (NGS) are used for the characterisation of miRNA. Proteogenomic approaches for detecting APP and novel miRNA profiling are essential in understanding the selective resistance to endotoxin in sheep. The results of these methods could help in understanding similar pathology in humans. It might also be helpful in the development of physiological and diagnostic screening assays for determining experimental inclusion and endpoints, and in clinical trials in future

Effect of surgical approach on bone vascularisation, fracture and soft tissue healing : comparison of less invasive to open approach

Over the past ten years, minimally invasive plate osteosynthesis (MIPO) for the fixation of long bone fractures has become a clinically accepted method with good outcomes, when compared to the conventional open surgical approach (open reduction internal fixation, ORIF). However, while MIPO offers some advantages over ORIF, it also has some significant drawbacks, such as a more demanding surgical technique and increased radiation exposure. No clinical or experimental study to date has shown a difference between the healing outcomes in fractures treated with the two surgical approaches. Therefore, a novel, standardised severe trauma model in sheep has been developed and validated in this project to examine the effect of the two surgical approaches on soft tissue and fracture healing. Twenty four sheep were subjected to severe soft tissue damage and a complex distal femur fracture. The fractures were initially stabilised with an external fixator. After five days of soft tissue recovery, internal fixation with a plate was applied, randomised to either MIPO or ORIF. Within the first fourteen days, the soft tissue damage was monitored locally with a compartment pressure sensor and systemically by blood tests. The fracture progress was assessed fortnightly by x-rays. The sheep were sacrificed in two groups after four and eight weeks, and CT scans and mechanical testing performed. Soft tissue monitoring showed significantly higher postoperative Creatine Kinase and Lactate Dehydrogenase values in the ORIF group compared to MIPO. After four weeks, the torsional stiffness was significantly higher in the MIPO group (p=0.018) compared to the ORIF group. The torsional strength also showed increased values for the MIPO technique (p=0.11). The measured total mineralised callus volumes were slightly higher in the ORIF group. However, a newly developed morphological callus bridging score showed significantly higher values for the MIPO technique (p=0.007), with a high correlation to the mechanical properties (R2=0.79). After eight weeks, the same trends continued, but without statistical significance. In summary, this clinically relevant study, using the newly developed severe trauma model in sheep, clearly demonstrates that the minimally invasive technique minimises additional soft tissue damage and improves fracture healing in the early stage compared to the open surgical approach method.

Agglutinins from marine macroalgae of the southeastern United States

Protein extracts from 22 species of marine macroalgae from Florida and North Carolina were compared for their abilities to agglutinate sheep and rabbit erythrocytes. Protein extracts from 21 algal species agglutinated rabbit erythrocytes compared to 19 for sheep erythrocytes. However, agglutination by brown algal extracts was variable. The agglutination produced by protein extracts from Dictyota dichotoma could be blocked by addition of polyvinylpyrrolidone. Protein extracts from North Carolina macroalgae were also tested against five bacterial species. Three of these agglutinated bacterial cells. Ulva curvata and Bryopsis plumosa agglutinated all five species. Protein extracts from five species of Florida algae were tested for their effects on mitogenesis in mouse splenocytes and human lymphocytes. Gracilaria tikvahiae HBOI Strain G-5, Ulva rigida and Gracilaria verrucosa HBOI Strain G-16S stimulated mitogenesis in mouse splenocytes, while Gracilaria tikvahiae HBOI Strain G-16stimulated mitogenesis in human lymphocytes.