The effect of rhBMP-2 on canine osteoblasts seeded onto 3D bioactive polycaprolactone scaffolds

Our strategy entails investigating the influence of varied concentrations (0, 10, 100 and 1000 ng/ml) of human recombinant bone morphogenetic protein-2 (rhBMP-2) on the osteogenic expression of canine osteoblasts, seeded onto poly-caprolactone 20% tricalcium phosphate (PCL-TCP) scaffolds in vitro. Biochemical assay revealed that groups with rhBMP-2 displayed an initial burst in cell growth that was not dose-dependent. However, after 13 days, cell growth declined to a value similar to control. Significantly less cell growth was observed for construct with 1000 ng/ml of rhBMP-2 from 20 days onwards. Confocal microscopy confirmed viability of osteoblasts and at day 20, groups seeded with rhBMP-2 displayed heightened cell death as compared to control. Phase contrast and scanning electron microscopy revealed that osteoblasts heavily colonized surfaces, rods and pores of the PCL-TCP scaffolds. This was consistent for all groups. Finally, Von Kossa and osteocalcin assays demonstrated that cells from all groups maintained their osteogenic phenotype throughout the experiment. Calcification was observed as early as four days after stimulation for groups seeded with rhBMP-2. In conclusion, rhBMP-2 seems to enhance the differentiated function of canine osteoblasts in a non-dose dependent manner. This resulted in accelerated mineralization, followed by death of osteoblasts as they underwent terminal differentiation. Notably, PCL-TCP scaffolds seeded only with canine osteoblasts could sustain excellent osteogenic expression in vitro. Hence, the synergy of PCL with bioactive TCP and rhBMP-2 in a novel composite scaffold, could offer an exciting approach for bone regeneration.

Immunohistochemical detection of survivin in canine lymphoma

Survivin is a member of the family of proteins known as 'inhibitors of apoptosis proteins'. Survivin has a role in cellular decisions concerning division and survival and is frequently expressed in neoplastic cells. The aim of the present study was to investigate immunohistochemically the expression of survivin in normal canine tissues and in canine lymphoma. A representative range of fetal and adult normal tissues as well as biopsy samples from dogs with lymphoma were assembled in tissue arrays. The lymphomas were classified according to the revised Kiel and to the Revised European American Lymphoma - World Health Organization (REAL-WHO) schemes. Polyclonal and monoclonal antisera cross-reactive with canine survivin identified cytoplasmic expression of the molecule in a broad range of normal canine cells. The same reagents demonstrated cytoplasmic labelling of more than 5% of cells in all 83 lymphoma samples tested with polyclonal antiserum and in 67 of 82 (82%) of samples tested with monoclonal antiserum. Survivin was expressed by a wide range of canine lymphoma subtypes, but the expression of this molecule in normal canine tissues must be considered if novel therapies targeting survivin are applied to the management of canine lymphoma. © 2010 Elsevier Ltd.

Evaluation of canine bone marrow-derived mesenchymal stem cells after long-term cryopreservation

Autologous bone marrow-derived mesenchymal stem cell (BMSCs)-based therapies show great potential in regenerative medicine. However, long-term storage and preservation of BMSCs for clinical use is still a great clinical challenge. The present study aimed to analyze the effect of long-term cryopreservation on the regenerative ability of BMSCs. After cryopreservation of BMSCs from beagle dogs for three years, cell viability, and quantitative analysis of alkaline phosphatase (ALP) activity, surface adherence, and mineralized nodule formation were analyzed. BMSCs in cell-scaffold complex were then implanted into nude mice. There was no significant difference in cell viability and ALP activity between osteogenic differentiation and non-osteogenic differentiation of BMSCs, and BMSCs in cell-scaffold complex retained osteogenic differentiation ability in vivo. These results indicate that long-term cryopreserved BMSCs maintain their have capacity to contribute to regeneration.

Experimental colonization of the canine urinary tract with the asymptomatic bacteriuria Escherichia coli strain 83972

Establishment of asymptomatic bacteriuria (ABU) with Escherichia coli 83972 is a viable prophylactic alternative to antibiotic therapy for the prevention of recurrent bacterial urinary tract infection in humans. Approximately 2 x 108 viable E. coli 83972 cells were introduced into the bladder of six healthy female dogs via a sterile urinary catheter. The presence of pyuria, depression, stranguria, pollakiuria and haematuria was documented for 6 weeks and urinalysis and aerobic bacterial cultures were performed every 24–72 h. Pyuria was present in all dogs on day 1 post-inoculation and 4/6 dogs (67%) had a positive urine culture on this day. Duration of colonization ranged from 0 to 10 days (median 4 days). Four dogs were re-inoculated on day 20. Duration of colonization following the second inoculation ranged from 1 to 3 days. No dog suffered pyrexia or appeared systemically unwell but all dogs initially exhibited mild pollakiuria and a small number displayed gross haematuria and/or stranguria. By day 3 of each trial all clinical signs had resolved. Persistent bacteriuria was not achieved in any dog but two dogs were colonized for 10 days following a single inoculation. Further research is required to determine whether establishment of ABU in dogs with recurrent urinary tract infection is a viable alternative to repeated doses of antimicrobial agents.

Histopathological features of bone regeneration in a canine segmental ulnar defect model

Background Today, finding an ideal biomaterial to treat the large bone defects, delayed unions and non-unions remains a challenge for orthopaedic surgeions and researchers. Several studies have been carried out on the subject of bone regeneration, each having its own advantages. The present study has been designed in vivo to evaluate the effects of cellular auto-transplantation of tail vertebrae on healing of experimental critical bone defect in a dog model. Methods Six indigenous breeds of dog with 32 ± 3.6 kg average weight from both sexes (5 males and 1 female) received bilateral critical-sized ulnar segmental defects. After determining the health condition, divided to 2 groups: The Group I were kept as control I (n = 1) while in Group II (experimental group; n = 5) bioactive bone implants were inserted. The defects were implanted with either autogeneic coccygeal bone grafts in dogs with 3-4 cm diaphyseal defects in the ulna. Defects were stabilized with internal plate fixation, and the control defects were not stabilized. Animals were euthanized at 16 weeks and analyzed by histopathology. Results Histological evaluation of this new bone at sixteen weeks postoperatively revealed primarily lamellar bone, with the formation of new cortices and normal-appearing marrow elements. And also reformation cortical compartment and reconstitution of marrow space were observed at the graft-host interface together with graft resorption and necrosis responses. Finally, our data were consistent with the osteoconducting function of the tail autograft. Conclusions Our results suggested that the tail vertebrae autograft seemed to be a new source of autogenous cortical bone in order to supporting segmental long bone defects in dogs. Furthermore, cellular autotransplantation was found to be a successful replacement for the tail vertebrae allograft bone at 3-4 cm segmental defects in the canine mid- ulna. Clinical application using graft expanders or bone autotransplantation should be used carefully and requires further investigation.

Malnutrition, gastroenteritis and trypsinogen concentration in hospitalised aboriginal children

Objective: To explore relationships between malnutrition and pancreatic damage in hospitalised aboriginal children. Methods: Immunoreactive trypsinogen (IRT) concentrations were measured in two populations of hospitalised aboriginal children in Australia; 472 children aged 0-3 years, in Alice Springs (Northern Territory); and 187 children aged 0-16 years in Mount Isa (Queensland). Correlation of whole blood IRT with height and weight z-scores, four-site skinfold thickness and upper arm circumference was sought. Results: In Mount Isa, the geometric mean IRT concentration rose with decreasing weight z-score. The IRT concentration was otherwise unrelated to nutritional indices. Sixty percent of the 39 Mount Isa patients with gastroenteritis and 24.5% of the 358 Alice Springs patients with gastroenteritis had an IRT concentration in the upper quartile for their population, compared with 16% for patients with other diagnoses in both populations. Conclusions: A high IRT concentration in patients with low weight z-scores is a confounding effect of gastroenteritis, and may result from subclinical pancreatic disease in gastroenteritis.

A controlled trial comparing the efficacy of rice-based and hypotonic glucose oral rehydration solutions in infants and young children with gastroenteritis

A prospective randomized trial was conducted to compare the efficacy of a rice-based oral rehydration solution (ORS) with glucose ORS in infants and children under 5 years of age with acute diarrhoea and mild to moderate dehydration (<10%). One hundred children presenting to a large metropolitan teaching hospital were eligible for entry to the study and were randomized to receive rice ORS or glucose ORS. Outcome measures were stool output (SO), duration of illness (DD) and recovery time to introduction of other fluids (RTF) and diet (RTD). Significant differences were found for all outcome measures in favour of the rice ORS group. Mean SO was lower (160 vs 213 mt; P<0.02), mean DD was reduced (17.3 vs 24.3 h; P = 0.03) and median RTF was decreased (12.7 vs 18.1 h; P< 0.001) in the rice ORS group compared with the glucose ORS group. The median rime to introduction of diet and mean length of hospital stay showed similar significant reductions. Our study has shown rice ORS to be an acceptable alternative to glucose ORS in young children and have shown that it is significantly more effective in reducing the course of diarrhoeal illness and the time taken to return to normal drinking and eating habits.

Canine-human transmission of Gastrospirillum hominis

We report electron microscopic evidence of transmission from a pet dog to a 12-year-girl of Gastrospirillum hominis which caused gastric disease in both that was eradicable with treatment. © 1994.

Comparison of two oral rehydration solutions in children with gastroenteritis in Australia

An open-label inpatient study is in progress to compare the efficacy and safety of two oral rehydration solutions in children and infants with acute diarrhea and mild to moderate dehydration. One solution (ORS-60) contains 60 mmol/L of sodium and 1.8% glucose, with a total osmolatity of 240 mosm/kg; the other (ORS-26) contains 26 mmol/L of sodium, 2.7% glucose, and 3.6% sucrose, with a total osmolality of 340 mosm/kg. An outcome analysis of 28 children with gastroenteritis indicated that ORS-60 (n = 13) reduced stool volume during the first eight hours after admission to a significantly greater (P < 0.05) extent than did ORS-26 (n = 15). Diarrhea had ceased by 24 hours in 64% of ORS-60 patients but in only 31% of ORS-26 patients, and the patients' clinical conidition was improved at eight hours in 84% of ORS-60 patients versus 60% of ORS-26 patients. Differences between treatments in degree of dehydration at each follow-up point, total duration of diarrhea, and duration of hospital stay were not detected. No adverse drug reactions occurred. Four patients received intravenous rehydration therapy, but none was considered a treatment failure. We conclude that the lower osmolar solution, ORS-60, conferred earlier recovey and reduced continuing fluid losses in the management of gastroenteritis.

Microbial risk from rainwater tanks in South East Queensland

Quantitative Microbial Risk Assessment (QMRA) analysis was used to quantify the risk of infection associated with the exposure to pathogens from potable and non-potable uses of roof-harvested rainwater in South East Queensland (SEQ). A total of 84 rainwater samples were analysed for the presence of faecal indicators (using culture based methods) and zoonotic bacterial and protozoan pathogens using binary and quantitative PCR (qPCR). The concentrations of Salmonella invA, and Giardia lamblia β-giradin genes ranged from 65-380 genomic units/1000 mL and 9-57 genomic units/1000 mL of water, respectively. After converting gene copies to cell/cyst number, the risk of infection from G. lamblia and Salmonella spp. associated with the use of rainwater for bi-weekly garden hosing was calculated to be below the threshold value of 1 extra infection per 10,000 persons per year. However, the estimated risk of infection from drinking the rainwater daily was 44-250 (for G. lamblia) and 85-520 (for Salmonella spp.) infections per 10,000 persons per year. Since this health risk seems higher than that expected from the reported incidences of gastroenteritis, the assumptions used to estimate these infection risks are critically discussed. Nevertheless, it would seem prudent to disinfect rainwater for potable use.

Pathogenicity of Plesiomonas shigelloides : interactions with eukaryotic host cells in vitro

Diarrhoea is one of the leading causes of morbidity and mortality in populations in developing countries and is a significant health issue throughout the world. Despite the frequency and the severity of the diarrhoeal disease, mechanisms of pathogenesis for many of the causative agents have been poorly characterised. Although implicated in a number of intestinal and extra-intestinal infections in humans, Plesiomonas shigelloides generally has been dismissed as an enteropathogen due to the lack of clearly demonstrated virulence-associated properties such as production of cytotoxins and enterotoxins or invasive abilities. However, evidence from a number of sources has indicated that this species may be the cause of a number of clinical infections. The work described in this thesis seeks to resolve this discrepancy by investigating the pathogenic potential of P. shigelloides using in vitro cell models. The focus of this research centres on how this organism interacts with human host cells in an experimental model. Very little is known about the pathogenic potential of P. shigel/oides and its mechanisms in human infections and disease. However, disease manifestations mimic those of other related microorganisms. Chapter 2 reviews microbial pathogenesis in general, with an emphasis on understanding the mechanisms resulting from infection with bacterial pathogens and the alterations in host cell biology. In addition, this review analyses the pathogenic status of a poorly-defined enteropathogen, P. shigelloides. Key stages of pathogenicity must occur in order for a bacterial pathogen to cause disease. Such stages include bacterial adherence to host tissue, bacterial entry into host tissues (usually required), multiplication within host tissues, evasion of host defence mechanisms and the causation of damage. In this study, these key strategies in infection and disease were sought to help assess the pathogenic potential of P. shigelloides (Chapter 3). Twelve isolates of P. shigelloides, obtained from clinical cases of gastroenteritis, were used to infect monolayers of human intestinal epithelial cells in vitro. Ultrastructural analysis demonstrated that P. shigelloides was able to adhere to the microvilli at the apical surface of the epithelial cells and also to the plasma membranes of both apical and basal surfaces. Furthermore, it was demonstrated that these isolates were able to enter intestinal epithelial cells. Internalised bacteria often were confined within vacuoles surrounded by single or multiple membranes. Observation of bacteria within membranebound vacuoles suggests that uptake of P. shigelloides into intestinal epithelial cells occurs via a process morphologically comparable to phagocytosis. Bacterial cells also were observed free in the host cell cytoplasm, indicating that P. shige/loides is able to escape from the surrounding vacuolar membrane and exist within the cytosol of the host. Plesiomonas shigelloides has not only been implicated in gastrointestinal infections, but also in a range of non-intestinal infections such as cholecystitis, proctitis, septicaemia and meningitis. The mechanisms by which P. shigelloides causes these infections are not understood. Previous research was unable to ascertain the pathogenic potential of P. shigel/oides using cells of non-intestinal origin (HEp-2 cells derived from a human larynx carcinoma and Hela cells derived from a cervical carcinoma). However, with the recent findings (from this study) that P. shigelloides can adhere to and enter intestinal cells, it was hypothesised, that P. shigel/oides would be able to enter Hela and HEp-2 cells. Six clinical isolates of P. shigelloides, which previously have been shown to be invasive to intestinally derived Caco-2 cells (Chapter 3) were used to study interactions with Hela and HEp-2 cells (Chapter 4). These isolates were shown to adhere to and enter both nonintestinal host cell lines. Plesiomonas shigelloides were observed within vacuoles surrounded by single and multiple membranes, as well as free in the host cell cytosol, similar to infection by P. shigelloides of Caco-2 cells. Comparisons of the number of bacteria adhered to and present intracellularly within Hela, HEp-2 and Caco-2 cells revealed a preference of P. shigelloides for Caco-2 cells. This study conclusively showed for the first time that P. shigelloides is able to enter HEp-2 and Hela cells, demonstrating the potential ability to cause an infection and/or disease of extra-intestinal sites in humans. Further high resolution ultrastructural analysis of the mechanisms involved in P. shigelloides adherence to intestinal epithelial cells (Chapter 5) revealed numerous prominent surface features which appeared to be involved in the binding of P. shige/loides to host cells. These surface structures varied in morphology from small bumps across the bacterial cell surface to much longer filaments. Evidence that flagella might play a role in bacterial adherence also was found. The hypothesis that filamentous appendages are morphologically expressed when in contact with host cells also was tested. Observations of bacteria free in the host cell cytosol suggests that P. shigelloides is able to lyse free from the initial vacuolar compartment. The vacuoles containing P. shigel/oides within host cells have not been characterised and the point at which P. shigelloides escapes from the surrounding vacuolar compartment has not been determined. A cytochemical detection assay for acid phosphatase, an enzymatic marker for lysosomes, was used to analyse the co-localisation of bacteria-containing vacuoles and acid phosphatase activity (Chapter 6). Acid phosphatase activity was not detected in these bacteria-containing vacuoles. However, the surface of many intracellular and extracellular bacteria demonstrated high levels of acid phosphatase activity, leading to the proposal of a new virulence factor for P. shigelloides. For many pathogens, the efficiency with which they adhere to and enter host cells is dependant upon the bacterial phase of growth. Such dependency reflects the timing of expression of particular virulence factors important for bacterial pathogenesis. In previous studies (Chapter 3 to Chapter 6), an overnight culture of P. shigelloides was used to investigate a number of interactions, however, it was unknown whether this allowed expression of bacterial factors to permit efficient P. shigelloides attachment and entry into human cells. In this study (Chapter 7), a number of clinical and environmental P. shigelloides isolates were investigated to determine whether adherence and entry into host cells in vitro was more efficient during exponential-phase or stationary-phase bacterial growth. An increase in the number of adherent and intracellular bacteria was demonstrated when bacteria were inoculated into host cell cultures in exponential phase cultures. This was demonstrated clearly for 3 out of 4 isolates examined. In addition, an increase in the morphological expression of filamentous appendages, a suggested virulence factor for P. shigel/oides, was observed for bacteria in exponential growth phase. These observations suggest that virulence determinants for P. shigel/oides may be more efficiently expressed when bacteria are in exponential growth phase. This study demonstrated also, for the first time, that environmental water isolates of P. shigelloides were able to adhere to and enter human intestinal cells in vitro. These isolates were seen to enter Caco-2 host cells through a process comparable to the clinical isolates examined. These findings support the hypothesis of a water transmission route for P. shigelloides infections. The results presented in this thesis contribute significantly to our understanding of the pathogenic mechanisms involved in P. shigelloides infections and disease. Several of the factors involved in P. shigelloides pathogenesis have homologues in other pathogens of the human intestine, namely Vibrio, Aeromonas, Salmonella, Shigella species and diarrhoeaassociated strains of Escherichia coli. This study emphasises the relevance of research into Plesiomonas as a means of furthering our understanding of bacterial virulence in general. As well it provides tantalising clues on normal and pathogenic host cell mechanisms.

Health risk from the use of roof-harvested rainwater in Southeast Queensland, Australia, as potable or nonpotable water, determined using quantitative microbial risk assessment

A total of 214 rainwater samples from 82 tanks were collected in urban Southeast Queensland (SEQ) in Australia and analysed for the zoonotic bacterial and protozoan pathogen using real-time binary PCR and quantitative PCR (qPCR). Quantitative Microbial Risk Assessment (QMRA) analysis was used to quantify the risk of infection associated with the exposure to potential pathogens from potable and non-potable uses of roof-harvested rainwater. Of the 214 samples tested, 10.7%, 9.8%, and 5.6%, and 0.4% samples were positive for Salmonella invA, Giardia lamblia β-giardin , Legionella pneumophila mip, and Campylobacter jejuni mapA genes. Cryptosporidium parvum could not be detected. The estimated numbers of viable Salmonella spp., G. lamblia β-giradin, and L. pneumophila genes ranged from 1.6 × 101 to 9.5 × 101 cells, 1.4 × 10-1 to 9.0 × 10-1 cysts, and 1.5 × 101 to 4.3 × 101 per 1000 ml of water, respectively. Six risk scenarios were considered from exposure to Salmonella spp., G. lamblia and L. pneumophila. For Salmonella spp., and G. lamblia, these scenarios were: (1) liquid ingestion due to drinking of rainwater on a daily basis (2) accidental liquid ingestion due to garden hosing twice a week (3) aerosol ingestion due to showering on a daily basis, and (4) aerosol ingestion due to hosing twice a week. For L. pneumophila, these scenarios were: (5) aerosol inhalation due to showering on a daily basis, and (6) aerosol inhalation due to hosing twice a week. The risk of infection from Salmonella spp., G. lamblia, and L. pneumophila associated with the use of rainwater for showering and garden hosing was calculated to be well below the threshold value of one extra infection per 10,000 persons per year in urban SEQ. However, the risk of infection from ingesting Salmonella spp. and G. lamblia via drinking exceeds this threshold value, and indicates that if undisinfected rainwater were ingested by drinking, then the gastrointestinal diseases of Salmonellosis and Giardiasis is expected to range from 5.0 × 100 to 2.8 × 101 (Salmonellosis) and 1.0 × 101 to 6.4 × 101 (Giardiasis) cases per 10,000 persons per year, respectively. Since this health risk seems higher than that expected from the reported incidences of gastroenteritis, the assumptions used to estimate these infection risks are critically examined. Nonetheless, it would seem prudent to disinfect rainwater for potable use.

Bayesian network for risk of diarrhea associated with the use of recycled water

Estimating potential health risks associated with recycled (reused) water is highly complex given the multiple factors affecting water quality. We take a conceptual model, which represents the factors and pathways by which recycled water may pose a risk of contracting gastroenteritis, convert the conceptual model to a Bayesian net, and quantify the model using one expert’s opinion. This allows us to make various predictions as to the risks posed under various scenarios. Bayesian nets provide an additional way of modeling the determinants of recycled water quality and elucidating their relative influence on a given disease outcome. The important contribution to Bayesian net methodology is that all model predictions, whether risk or relative risk estimates, are expressed as credible intervals.

SNP diversity of Enterococcus faecalis and Enterococcus faecium in a South East Queensland waterway, Australia, and associated antibiotic resistance gene profiles

BACKGROUND: Enterococcus faecalis and Enterococcus faecium are associated with faecal pollution of water, linked to swimmer-associated gastroenteritis and demonstrate a wide range of antibiotic resistance. The Coomera River is a main water source for the Pimpama-Coomera watershed and is located in South East Queensland, Australia, which is used intensively for agriculture and recreational purposes. This study investigated the diversity of E. faecalis and E. faecium using Single Nucleotide Polymorphisms (SNPs) and associated antibiotic resistance profiles. RESULTS: Total enterococcal counts (cfu/ml) for three/six sampling sites were above the United States Environmental Protection Agency (USEPA) recommended level during rainfall periods and fall into categories B and C of the Australian National Health and Medical Research Council (NHMRC) guidelines (with a 1-10% gastrointestinal illness risk). E. faecalis and E. faecium isolates were grouped into 29 and 23 SNP profiles (validated by MLST analysis) respectively. This study showed the high diversity of E. faecalis and E. faecium over a period of two years and both human-related and human-specific SNP profiles were identified. 81.8% of E. faecalis and 70.21% of E. faecium SNP profiles were associated with genotypic and phenotypic antibiotic resistance. Gentamicin resistance was higher in E. faecalis (47% resistant) and harboured the aac(6')-aph(2') gene. Ciprofloxacin resistance was more common in E. faecium (12.7% resistant) and gyrA gene mutations were detected in these isolates. Tetracycline resistance was less common in both species while tet(L) and tet(M) genes were more prevalent. Ampicillin resistance was only found in E. faecium isolates with mutations in the pbp5 gene. Vancomycin resistance was not detected in any of the isolates. We found that antibiotic resistance profiles further sub-divided the SNP profiles of both E. faecalis and E. faecium. CONCLUSIONS: The distribution of E. faecalis and E. faecium genotypes is highly diverse in the Coomera River. The SNP genotyping method is rapid and robust and can be applied to study the diversity of E. faecalis and E. faecium in waterways. It can also be used to test for human-related and human-specific enterococci in water. The resolving power can be increased by including antibiotic-resistant profiles which can be used as a possible source tracking tool. This warrants further investigation.