Isolation of Pseudomonas pseudomallei from clay layers at defined depths

Twelve strains of Pseudomonas pseudomallei were isolated from the soil and water of a sheep paddock over a two-year period. The organism was recovered from the clay layer of the soil profile as well as from water that seeps into this layer during the "wet" season. Five isolates were obtained before the commencement of the "wet" season; environmental factors appear to play an important role in the survival of Ps. pseudomallei during the "dry" season. Lower isolation rates were recorded than those indicated by workers in southeast Asia and Iran.

Detection of Chlamydiaceae DNA in veterinary specimens using a family-specific PCR

Aims: The aim of this work was to develop a rapid molecular test for the detection of the Chlamydiaceae family, irrespective of the species or animal host. Methods and Results: The method described herein is a polymerase chain reaction targeting the 16S rRNA gene of the Chlamydiaceae family, and the results demonstrate that the test reacts with five reference Chlamydiaceae but none of the 19 other bacterial species or five uninfected animal tissues tested. The results also indicate the enhanced sensitivity of this test when compared with conventional culture or serology techniques. This is demonstrated through parallel testing of six real clinical veterinary cases and confirmatory DNA sequence analysis. Conclusions, Significance and Impact of the Study: This test can be used by veterinary diagnostic laboratories for rapid detection of Chlamydiaceae in veterinary specimens, with no restriction of chlamydial species or animal host. The test does not differentiate chlamydial species, and if required, speciation must be carried out retrospectively using alternate methods. However, for the purpose of prescribing therapy for chlamydiosis, this test would be an invaluable laboratory tool.

Attributes of clinical role models as described by senior veterinary students in Australia

Role models incite admiration and provide inspiration, contributing to learning as students aspire to emulate their example. The attributes of physician role models for medical trainees are well documented, but they remain largely unexplored in the context of veterinary medical training. The aim of the current study was to describe the attributes that final-year veterinary students (N=213) at the University of Queensland identified when reflecting on their clinical role models. Clinical role model descriptions provided by students were analyzed using concept-mapping software (Leximancer v. 2.25). The most frequent and highly connected concepts used by students when describing their role model(s) included clients, vet, and animal. Role models were described as good communicators who were skilled at managing relationships with clients, patients, and staff. They had exemplary knowledge, skills, and abilities, and they were methodical and conducted well-structured consultations. They were well respected and, in turn, demonstrated respect for clients, colleagues, staff, and students alike. They were also good teachers and able to tailor explanations to suit both clients and students. Findings from this study may serve to assist with faculty development and as a basis for further research in this area.

Rapid typing of P multocida

Fowl cholera, caused by P. multocida, is a serious disease of poultry with sudden surges in mortality and an emerging disease of the free ranged poultry industries. This project will develop a more rapid and cost effective screening method for P. multocida. The impacts of this new method are manifold: It will lead to an improved understanding of the epidemiology of fowl cholera and the possible sources of entry onto the farm leading to improved biosecurity measures and control programs. Another impact is improved serotyping, which will ensure more effective and targeted vaccination programs. Improving prevention and control programs and decreasing the reliance on antibiotics will enhance the sustainability and profitability of the industry.

The epidemiology of severe Streptococcus pyogenes disease in Europe

Diseases caused by the Lancefield group A streptococcus, Streptococcus pyogenes, are amongst the most challenging to clinicians and public health specialists alike. Although severe infections caused by S. pyogenes are relatively uncommon, affecting around 3 per 100,000 of the population per annum in developed countries, the case fatality is high relative to many other infections. Despite a long scientific tradition of studying their occurrence and characteristics, many aspects of their epidemiology remain poorly understood, and potential control measures undefined. Epidemiological studies can play an important role in identifying host, pathogen and environmental factors associated with risk of disease, manifestation of particular syndromes or poor survival. This can be of value in targeting prevention activities, as well directing further basic research, potentially paving the way for the identification of novel therapeutic targets. The formation of a European network, Strep-EURO, provided an opportunity to explore epidemiological patterns across Europe. Funded by the Fifth Framework Programme of the European Commission s Directorate-General for Research (QLK2.CT.2002.01398), the Strep-EURO network was launched in September 2002. Twelve participants across eleven countries took part, led by the University of Lund in Sweden. Cases were defined as patients with S. pyogenes isolated from a normally sterile site, or non-sterile site in combination with clinical signs of streptococcal toxic shock syndrome (STSS). All participating countries undertook prospective enhanced surveillance between 1st January 2003 and 31st December 2004 to identify cases diagnosed during this period. A standardised surveillance dataset was defined, comprising demographic, clinical and risk factor information collected through a questionnaire. Isolates were collected by the national reference laboratories and characterised according to their M protein using conventional serological and emm gene typing. Descriptive statistics and multivariable analyses were undertaken to compare characteristics of cases between countries and identify factors associated with increased risk of death or development of STSS. Crude and age-adjusted rates of infection were calculated for each country where a catchment population could be defined. The project succeeded in establishing the first European surveillance network for severe S. pyogenes infections, with 5522 cases identified over the two years. Analysis of data gathered in the eleven countries yielded important new information on the epidemiology of severe S. pyogenes infections in Europe during the 2000s. Comprehensive epidemiological data on these infections were obtained for the first time from France, Greece and Romania. Incidence estimates identified a general north-south gradient, from high to low. Remarkably similar age-standardised rates were observed among the three Nordic participants, between 2.2 and 2.3 per 100,000 population. Rates in the UK were higher still, 2.9/100,000, elevated by an upsurge in drug injectors. Rates from these northern countries were reasonably close to those observed in the USA and Australia during this period. In contrast, rates of reports in the more central and southern countries (Czech Republic, Romania, Cyprus and Italy) were substantially lower, 0.3 to 1.5 per 100,000 population, a likely reflection of poorer uptake of microbiological diagnostic methods within these countries. Analysis of project data brought some new insights into risk factors for severe S. pyogenes infection, especially the importance of injecting drug users in the UK, with infections in this group fundamentally reshaping the epidemiology of these infections during this period. Several novel findings arose through this work, including the high degree of congruence in seasonal patterns between countries and the seasonal changes in case fatality rates. Elderly patients, those with compromised immune systems, those who developed STSS and those infected with an emm/M78, emm/M5, emm/M3 or emm/M1 were found to be most likely to die as a result of their infection, whereas those diagnosed with cellulitis, septic arthritis, puerperal sepsis or with non-focal infection were associated with low risk of death, as were infections occurring during October. Analysis of augmented data from the UK found use of NSAIDs to be significantly associated with development of STSS, adding further fuel to the debate surrounding the role of NSAIDs in the development of severe disease. As a largely community-acquired infection, occurring sporadically and diffusely throughout the population, opportunities for control of severe infections caused by S. pyogenes remain limited, primarily involving contact chemoprophylaxis where clusters arise. Analysis of UK Strep-EURO data were used to quantify the risk to household contacts of cases, forming the basis of national guidance on the management of infection. Vaccines currently under development could offer a more effective control programme in future. Surveillance of invasive infections caused by S. pyogenes is of considerable public health importance as a means of identifying long and short-term trends in incidence, allowing the need for, or impact of, public health measures to be evaluated. As a dynamic pathogen co-existing among a dynamic population, new opportunities for exploitation of its human host are likely to arise periodically, and as such continued monitoring remains essential.

Epidemiology and management of tobacco streak virus in sunflower and pulse crops

Epidemiology and management of tobacco streak virus in sunflower and pulse crops.

Studies of the genetic epidemiology of cardiovascular disease: focus on inflammatory candidate genes

Cardiovascular disease (CVD) is a complex disease with multifactorial aetiology. Both genetic and environmental factors contribute to the disease risk. The lifetime risk for CVD differs markedly between men and women, men being at increased risk. Inflammatory reaction contributes to the development of the disease by promoting atherosclerosis in artery walls. In the first part of this thesis, we identified several inflammatory related CVD risk factors associating with the amount of DNA from whole blood samples, indicating a potential source of bias if a genetic study selects the participants based on the available amount of DNA. In the following studies, this observation was taken into account by applying whole genome amplification to samples otherwise subjected to exclusion due to very low DNA yield. We continued by investigating the contribution of inflammatory genes to the risk for CVD separately in men and women, and looked for sex-genotype interaction. In the second part, we explored a new candidate gene and its role in the risk for CVD. Selenoprotein S (SEPS1) is a membrane protein residing in the endoplasmic reticulum where it participates in retro-translocation of unfolded proteins to cytosolic protein degradation. Previous studies have indicated that SEPS1 protects cells from oxidative stress and that variations in the gene are associated with circulating levels of inflammatory cytokines. In our study, we identified two variants in the SEPS1 gene, which associated with coronary heart disease and ischemic stroke in women. This is, to our knowledge, the first study suggesting a role of SEPS1 in the risk for CVD after extensively examining the variation within the gene region. In the third part of this thesis, we focused on a set of seven genes (angiotensin converting enzyme, angiotensin II receptor type I, C-reactive protein (CRP), and fibrinogen alpha-, beta-, and gamma-chains (FGA, FGB, FGG)) related to inflammatory cytokine interleukin 6 (IL6) and their association with the risk for CVD. We identified one variant in the IL6 gene conferring risk for CVD in men and a variant pair from IL6 and FGA genes associated with decreased risk. Moreover, we identified and confirmed an association between a rare variant in the CRP gene and lower CRP levels, and found two variants in the FGA and FGG genes associating with fibrinogen. The results from this third study suggest a role for the interleukin 6 pathway genes in the pathogenesis of CVD and warrant further studies in other populations. In addition to the IL6 -related genes, we describe in this thesis several sex-specific associations in other genes included in this study. The majority of the findings were evident only in women encouraging other studies of cardiovascular disease to include and analyse women separately from men.

Human Torque teno virus: epidemiology, cell biology and immunology

Torque teno virus (TTV) was discovered in 1997 in the serum of a Japanese patient who had a post-transfusion hepatitis of unknown etiology. It is a small virus containing a circular single-stranded DNA genome which is unique among human viruses. Within a few years after its discovery, the TTVs were noted to form a large family of viruses with numerous genotypes. TTV is highly prevalent among the general population throughout the world, and persistent infections and co-infections with several genotypes occur frequently. However, the pathogenicity and the mechanism for the sustained occurrence of the virus in blood are at present unclear. To determine the prevalence of TTV in Finland, we set up PCR methods and examined the sera of asymptomatic subjects for the presence of TTV DNA and for genotype-6 DNA. TTV was found to be highly prevalent also in Finland; 85% of adults harbored TTV in their blood, and 4% were infected with genotype-6. In addition, TTV DNA was detected in a number of different tissues, with no tissue-type or symptom specificity. Most cell-biological events during TTV infections are at the moment unknown. Replicating TTV DNA has, however, been detected in liver and the hematopoietic compartment, and three mRNAs are known to be generated. To characterize TTV cell biology in more detail, we cloned in full length the genome of TTV genotype 6. We showed that in human kidney-derived cells TTV produces altogether six proteins with distinct subcellular localizations. TTV mRNA transcription was detected in all cell lines transfected with the full-length clone, and TTV DNA replicated in several of them, including those of erythroid, kidney, and hepatic origin. Furthermore, the viral DNA replication was shown to utilize the cellular DNA polymerases. Diagnoses of TTV infections have been based almost solely on PCR, whereas serological tests, measuring antibody responses, would give more information on many aspects of these infections. To investigate the TTV immunology in more detail, we produced all six TTV proteins for use as antigens in serological tests. We detected in human sera IgM and IgG antibodies to occur simultaneously with TTV DNA, and observed appearance of TTV DNA regardless of pre-existing antibodies, and disappearance of TTV DNA after antibody appearance. The genotype-6 nucleotide sequence remained stable for years within the infected subjects, suggesting that some mechanism other than mutations is used by this minute virus to evade our immune system and to establish chronic infections in immunocompetent subjects.

Dengue virus infection : Diagnostics and molecular epidemiology

Dengue is a mosquito-borne viral disease caused by the four dengue virus serotypes (DENV-1-4) and is currently considered as the most important arthropod-borne viral disease in the world. Nearly half of the human population lives in risk areas, and 50-100 million infections occur yearly according to World Health Organization. The disease can vary from a mild febrile disease to severe haemorrhagic fever and shock. A secondary infection with heterologous serotype increases the risk for severe disease outcome. During the last three decades the impact of dengue has dramatically increased in the endemic areas including the tropics and subtropics of the world. The current situation with massive epidemics of severe disease forms has been associated with socio-ecological changes that have increased the transmission and enabled the co-circulation of different serotypes. Consequently, an increase of dengue has also been observed in travelers visiting these areas. Currently approximately 30 cases are diagnosed yearly in Finnish travelers. In travelers dengue is rarely a life-threatening disease, however in the current study, a fatality was documented in a young Finnish patient who experienced a prolonged primary dengue infection. To improve particularly early laboratory diagnostics, a novel real-time RT-PCR method was developed for the detection of DENV-1-4 RNA based on TaqMan chemistry. The method was shown to be sensitive and specific for detecting DENV RNA and suitable for diagnostic use. The newly developed real-time RT-PCR was compared to other available early diagnostic methods including IgM and NS1 antigen detection using a panel of selected patient samples. The results suggest that the best diagnostic rates are achieved by a combination of IgM with RNA or NS1 detection. The dengue virus strains studied here included the first DENV strains isolated from serum samples of Finnish travelers collected in 2000-2005. The results of sequence analysis demonstrated that the 11 isolates included all four DENV serotypes and presented a global sample of DENV strains from different geographical areas including Asia, Africa and South America. In the present study sequence analysis was also carried out for a collection of 23 novel DENV-2 isolates from Venezuelan patients collected in 1999-2005. The Venezuelan DENV-2 exclusively represented the American-Asian genotype, suggesting that no foreign DENV-2 lineages have recently been introduced to the country. The results also suggest that the DENV-2 viruses detected earlier from Venezuela have been maintained in the area where they have evolved into several lineages. This is in contrast to the pattern observed in some other dengue endemic areas, where introductions of novel virus types and lineages are frequently detected.

Foodborne Yersinia : Identification and Molecular Epidemiology of Isolates from Human Infections

Yersinia enterocolitica and Yersinia pseudotuberculosis are among the major enteropathogenic bacteria causing infections in humans in many industrialized countries. In Finland, Y. pseudotuberculosis has caused 10 outbreaks among humans during 1997-2008. Some of these outbreaks have been very extensive involving over 400 cases; mainly children attending schools and day-care. Y. enterocolitica, on the contrary, has caused mainly a large number of sporadic human infections in Finland. Y. pseudotuberculosis is widespread in nature, causing infections in a variety of domestic and wild animals. Foodborne transmission of human infections has long been suspected, however, attempts to trace the pathogen have been unsuccessful before this study that epidemiologically linked Y. pseudotuberculosis to a specific food item. Furthermore, due to modern food distribution systems, foodborne outbreaks usually involve many geographically separate infection clusters difficult to identify as part of the same outbreak. Among pathogenic Y. enterocolitica, the global predominance of one genetically homogeneous type (bioserotype 4/O:3) is a challenge to the development of genetic typing methods discriminatory enough for epidemiological purposes, for example, for tracing back to the sources of infections. Furthermore, the diagnostics of Y. enterocolitica infections is hampered because clinical laboratories easily misidentify some other members of the Yersinia species (Y. enterocolitica–like species) as Y. enterocolitica. This results in misleading information on the prevalence and clinical significance of various Yersinia isolates. The aim of this study was to develop and optimize molecular typing methods to be used in epidemiological investigations of Y. enterocolitica and Y. pseudotuberculosis, particularly in active surveillance and outbreak investigations of Y. pseudotuberculosis isolates. The aim was also to develop a simplified set of phenotypic tests that could be used in routine diagnostic laboratories for the correct identification of Y. enterocolitica and Y. enterocolitica –like species. A PFGE method designed here for typing of Y. pseudotuberculosis was efficient in linking the geographically dispersed and apparently unrelated Y. pseudotuberculosis infections as parts of the same outbreak. It proved to be useful in active laboratory-based surveillance of Y. pseudotuberculosis outbreaks. Throughout the study period, information about the diversity of genotypes among outbreak and non-outbreak related strains of human origin was obtained. Also, to our knowledge, this was the first study to epidemiologically link a Y. pseudotuberculosis outbreak of human illnesses to a specific food item, iceberg lettuce. A novel epidemiological typing method based on the use of a repeated genomic region (YeO:3RS) as a probe was developed for the detection and differentiation between strains of Y. enterocolitica subspecies palearctica. This method was able to increase the discrimination in a set of 106 previously PFGE typed Finnish Y. enterocolitica bioserotype 4/O:3 strains among which two main PFGE genotypes had prevailed. The developed simplified method was a more reliable tool than the commercially available biochemical test kits for differentiation between Y. enterocolitica and Y. enterocolitica –like species. In Finland, the methods developed for Y. enterocolitica and Y. pseudotuberculosis have been used to improve the identification protocols and in subsequent outbreak investigations.