Detection of African Swine Fever Virus Antibodies in Serum and Oral Fluid Specimens Using a Recombinant Protein 30 (p30) Dual Matrix Indirect ELISA

In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence.

African swine fever virus transmission cycles in Central Europe: evaluation of wild boar-soft tick contacts through detection of antibodies against Ornithodoros erraticus saliva antigen.

BACKGROUND African swine fever (ASF) is one of the most complex viral diseases affecting both domestic and wild pigs. It is caused by ASF virus (ASFV), the only DNA virus which can be efficiently transmitted by an arthropod vector, soft ticks of the genus Ornithodoros. These ticks can be part of ASFV-transmission cycles, and in Europe, O. erraticus was shown to be responsible for long-term maintenance of ASFV in Spain and Portugal. In 2014, the disease has been reintroduced into the European Union, affecting domestic pigs and, importantly, also the Eurasian wild boar population. In a first attempt to assess the risk of a tick-wild boar transmission cycle in Central Europe that would further complicate eradication of the disease, over 700 pre-existing serum samples from wild boar hunted in four representative German Federal States were investigated for the presence of antibodies directed against salivary antigen of Ornithodoros erraticus ticks using an indirect ELISA format. RESULTS Out of these samples, 16 reacted with moderate to high optical densities that could be indicative of tick bites in sampled wild boar. However, these samples did not show a spatial clustering (they were collected from distant geographical regions) and were of bad quality (hemolysis/impurities). Furthermore, all positive samples came from areas with suboptimal climate for soft ticks. For this reason, false positive reactions are likely. CONCLUSION In conclusion, the study did not provide stringent evidence for soft tick-wild boar contact in the investigated German Federal States and thus, a relevant involvement in the epidemiology of ASF in German wild boar is unlikely. This fact would facilitate the eradication of ASF in the area, although other complex relations (wild boar biology and interactions with domestic pigs) need to be considered.

Evaluation of the risk factors contributing to the African swine fever occurrence in Sardinia, Italy

This study assesses the relation between hypothesized risk factors and African swine fever virus (ASFV) distribution in Sardinia (Italy) after the beginning of the eradication program in 1993, using a Bayesian multivariable logistic regression mixed model. Results indicate that the probability of ASFV occurrence in Sardinia was associated to particular socio-cultural, productive and economical factors found in the region, particularly to large number of confined (i.e., closed) farms (most of them backyard), high road density, high mean altitude, large number of open fattening farms, and large number of pigs per commune. Conversely, large proportion of open farms with at least one census and large proportion of open farms per commune, were found to be protective factors for ASFV. Results suggest that basic preventive and control strategies, such as yearly census or registration of the pigs per farm and better control of the public lands where pigs are usually raised, together with endanced effords of outreach and communication with pig producers should help in the success of the eradication program for ASF in the Island. Methods and results presented here will inform decision making to better control and eradicate ASF in Sardinia and in all those areas with similar management and epidemiological conditions.

Modular framework to assess the risk of African swine fever virus entry into the European Union

BACKGROUND The recent occurrence and spread of African swine fever (ASF) in Eastern Europe is perceived as a serious risk for the pig industry in the European Union (EU). In order to estimate the potential risk of ASF virus (ASFV) entering the EU, several pathways of introduction were previously assessed separately. The present work aimed to integrate five of these assessments (legal imports of pigs, legal imports of products, illegal imports of products, fomites associated with transport and wild boar movements) into a modular tool that facilitates the visualization and comprehension of the relative risk of ASFV introduction into the EU by each analyzed pathway. RESULTS The framework's results indicate that 48% of EU countries are at relatively high risk (risk score 4 or 5 out of 5) for ASFV entry for at least one analyzed pathway. Four of these countries obtained the maximum risk score for one pathway: Bulgaria for legally imported products during the high risk period (HRP); Finland for wild boar; Slovenia and Sweden for legally imported pigs during the HRP. Distribution of risk considerably differed from one pathway to another; for some pathways, the risk was concentrated in a few countries (e.g., transport fomites), whereas other pathways incurred a high risk for 4 or 5 countries (legal pigs, illegal imports and wild boar). CONCLUSIONS The modular framework, developed to estimate the risk of ASFV entry into the EU, is available in a public domain, and is a transparent, easy-to-interpret tool that can be updated and adapted if required. The model's results determine the EU countries at higher risk for each ASFV introduction route, and provide a useful basis to develop a global coordinated program to improve ASFV prevention in the EU.

Risk of African swine fever introduction into the European Union through transport-associated routes: returning trucks and waste from international ships and planes

BACKGROUND The uncontrolled presence of African swine fever (ASF) in Russian Federation (RF) poses a serious risk to the whole European Union (EU) pig industry. Although trade of pigs and their products is banned since the official notification in June 2007, the potential introduction of ASF virus (ASFV) may occur by other routes, which are very frequent in ASF, and more difficult to control, such as contaminated waste or infected vehicles. This study was intended to estimate the risk of ASFV introduction into the EU through three types of transport routes: returning trucks, waste from international ships and waste from international planes, which will be referred here as transport-associated routes (TAR). Since no detailed and official information was available for these routes, a semi-quantitative model based on the weighted combination of risk factors was developed to estimate the risk of ASFV introduction by TAR. Relative weights for combination of different risk factors as well as validation of the model results were obtained by an expert opinion elicitation. RESULTS Model results indicate that the relative risk for ASFV introduction through TAR in most of the EU countries (16) is low, although some countries, specifically Poland and Lithuania, concentrate high levels of risk, the returning trucks route being the analyzed TAR that currently poses the highest risk for ASFV introduction into the EU. The spatial distribution of the risk of ASFV introduction varies importantly between the analyzed introduction routes. Results also highlight the need to increase the awareness and precautions for ASF prevention, particularly ensuring truck disinfection, to minimize the potential risk of entrance into the EU. CONCLUSIONS This study presents the first assessment of ASF introduction into the EU through TAR. The innovative model developed here could be used in data scarce situations for estimating the relative risk associated to each EU country. This simple methodology provides a rapid and easy to interpret results on risk that may be used for a target and cost-effective allocation of resources to prevent disease introduction.

Introduction of African Swine Fever into the European Union through Illegal Importation of Pork and Pork Products

Transboundary animal diseases can have very severe socio-economic impacts when introduced into new regions. The history of disease incursions into the European Union suggests that initial outbreaks were often initiated by illegal importation of meat and derived products. The European Union would benefit from decision-support tools to evaluate the risk of disease introduction caused by illegal imports in order to inform its surveillance strategy. However, due to the difficulty in quantifying illegal movements of animal products, very few studies of this type have been conducted. Using African swine fever as an example, this work presents a novel risk assessment framework for disease introduction into the European Union through illegal importation of meat and products. It uses a semi-quantitative approach based on factors that likely influence the likelihood of release of contaminated smuggled meat and products, and subsequent exposure of the susceptible population. The results suggest that the European Union is at non-negligible risk of African swine fever.

2016 Dengue Fever Virus Cases

Dengue fever is the most common cause of fever in travelers returning from the Caribbean, Central America, and South Central Asia.* Dengue infections are commonly reported from most tropical countries of the South Pacific, Asia, the Caribbean, the Americas, and Africa. This disease is caused by four similar viruses (DENV-1, -2, -3, and -4) and is spread through the bites of infected mosquitoes. For information on current outbreaks, consult CDC’s Travelers’ Health website (http://www.cdc.gov/travel). Dengue fever is a severe, flu-like illness that affects infants, young children and adults, but seldom causes death. Dengue should be suspected when a high fever (40°C/104°F) is accompanied by two of the following symptoms: severe headache, pain behind the eyes, muscle and joint pains, nausea, vomiting, swollen glands or rash. Symptoms usually last for 2–7 days, after an incubation period of 4–10 days following the bite from an infected mosquito.

The impact and control of malignant catarrhal fever in Tanzania

Malignant Catarrhal Fever (MCF), an often-lethal infectious disease, presents as a variable complex of lesions in susceptible ungulate species. The disease is caused by a -herpesvirus following transmission from an inapparent carrier host. Two major epidemiological forms exist: wildebeest-associated MCF (WA-MCF), in which the virus is transmitted to susceptible species by wildebeest calves less than approximately four months of age, and sheepassociated MCF (SA-MCF) in which the virus is spread by sheep (primarily adolescents). Due to the lack of an in-vitro propagation system for the causative agent of the more economically significant SA-MCF, and with the expectation that cross-protective immunity may be provided, vaccine development has focused on the more easily propagated alcelaphine herpesvirus-1 (AlHV-1) that causes WA-MCF. In 2008 a direct viral challenge trial showed that a novel vaccine, employing an attenuated AlHV-1 (atAlHV-1) `C5000 virus strain, protected British Friesian-Holstein (FH) cattle against an intranasal challenge with virulent AlHV-1 `C5000 virus. For cattle keeping people living near wildebeest calving areas in sub-Saharan Africa an effective vaccine would have value as it would release them from the costly annual disease avoidance strategy of having to move their herds away from the oncoming wildebeest. On the other hand, an effective vaccine will release herd owners from the need to avoid MCF, allowing them to graze their cattle alongside wildebeest on the highly nutritious pastures of the calving areas. As such conservationists have raised concerns that the development of a vaccine might lead to detrimental grazing competition. The principle objective of this study was to test the novel vaccine on Tanzanian shorthorn zebu cross cattle (SZC).We did this firstly using a natural challenge field trial (Chapter Two) which demonstrated that immunisation with the atAlHV-1 vaccine was well tolerated and induced an oro-nasopharyngeal AlHV-1-specific and -neutralising antibody response. This resulted in an immunity in SZC cattle that was partially protective and reduced naturally transmitted infection by 56%. We also demonstrated that non-fatal infections occurred with a much higher frequency than previously thought. Because the calculated efficacy of the vaccine was less than that seen in British FH cattle we wanted to determine whether host factors, particular to SZC cattle, had impacted the outcomes of the field trial. To do this we repeated the 2008 direct viral challenge trial using SZC cattle (Chapter Four). During this trial we also investigated whether the recombinant bacterial flagellin monomer (FliC), when used as an adjuvant, might improve the vaccine’s efficacy. The findings from this trial indicated that direct challenge with pathogenic AlHV-1 is effective at inducing MCF in SZC cattle and that FliC is not an appropriate adjuvant for this vaccine. Furthermore, with less control group cattle dying of MCF than expected we speculate that SZC cattle may have a degree of resistance to MCF that affords them protection from infection and developing fatal disease. In Chapter Three we investigated aspects of the epidemiology of MCF, specifically whether wildebeest placenta, long implicated by Maasai cattle owners as a source of MCF, might play a role in viral transmission. Additionally, through comparative sequence analysis, at two specific genes (A9.5 and ORF50) of wild-type and atAlHV-1, we investigated whether the `C5000 strain, the source of which was taken from Africa more than 40 years ago, was appropriate for vaccine development. The detection of AlHV-1 virus in approximately 50% of placentae indicated that infection can occur in-utero and that this tissue might play a role in disease transmission. And, despite describing three new alleles of the A9.5 gene (supporting previous evidence that this gene is polymorphic and encodes a secretory protein with interleukin-4 as the major homologue), the observation that the most frequently detected haplotypes, in both wild-type and attenuated AlHV-1, were identical suggests that AlHV-1 has a slow molecular clock and that the attenuated strain was appropriate for vaccine development. In Chapter Five we present the first quantitative assessment of the annual MCF avoidance costs that Maasai pastoralists incur. In particular we estimated that as a result of MCF avoidance 64% of the total daily milk yield during the MCF season was not available to be used by the 81% of the family unit remaining at the permanent boma. This represents an upper-bound loss of approximately 8% of a household0s annual income. Despite these considerable losses we concluded that, given an incidence of fatal MCF in cattle living in wildebeest calving areas of 5% to 10%, if herd owners were to stop trying to avoid MCF by allowing their cattle to graze alongside wildebeest, any gains made through increased availability of milk, improved body condition and reduced energy demands would be offset by an increase in MCF-incidence. With the development of an effective vaccine, however, this alternative strategy might become optimal. The overall conclusion we draw therefore is that, despite the substantial costs incurred each year avoiding MCF, the partial protection afforded by the novel vaccine strategy is not sufficient to warrant a wholesale change in disease avoidance strategy. Nonetheless, even the partial protection provided by this vaccine could be of value to protect animals that cannot be moved, for example where some of the herd remain at the boma to provide milk or where land-use changes make traditional disease avoidance difficult. Furthermore, the vaccine may offer a feasible solution to some of the current land-use challenges and conflicts, providing a degree of protection to valuable livestock where avoidance strategies are not possible, but with less risk of precipitating the potentially damaging environmental consequences, such as overgrazing of highly nutritious seasonal pastures, that might result if herd owners decide they no longer need to avoid wildebeest.

Diagnóstico molecular de tristeza parasitária bovina em rhipicephalus microplus da região tropical do méxico / molecular diagnosis of cattle tick fever agents in rhipicephalus microplus from tropical region of mexico

2016

Increased body temperature following subarachnoid haemorrhage : a retrospective correlational study

Introduction: Nursing clinicians are primarily responsible for the monitoring and treatment of increased body temperature. The body temperature of patients during their acute care hospital stay is measured at regular repeated intervals. In the event a patient is assessed with an elevated temperature, a multitude of decisions are required. The action of instigating temperature reducing strategies is based upon the assumption that elevated temperature is harmful and that the strategy employed will have some beneficial effect. Background and Significance: The potential harmful effects of increased body temperature (fever, hyperthermia) following neurological insult are well recognised. Although few studies have investigated this phenomenon in the diagnostic population of non-traumatic subarachnoid haemorrhage, it has been demonstrated that increased body temperature occurs in 41 to 72% of patients with poor clinical outcome. However, in the Australian context the frequency, or other characteristics of increased body temperature, as well as the association between increased body temperature with poor clinical outcome has not been established. Design: This study used a correlational study design to: describe the frequency, duration and timing of increased body temperature; determine the association between increased body temperature and clinical outcome; and describe the clinical interventions used to manage increased body temperature in patients with non-traumatic subarachnoid haemorrhage. A retrospective clinical chart audit was conducted on 43 patients who met the inclusion criteria. Findings: The major findings of this study were: increased body temperature occurred frequently; persisted for a long time; and onset did not occur until 20 hours after primary insult; increased body temperature was associated with death or dependent outcome; and no intervention was recorded in many instances. Conclusion: This study has quantified in a non-traumatic subarachnoid haemorrhage patient population the characteristics of increased body temperature, established an association between increased body temperature with death or dependent outcome and described the current management of elevated temperatures in the Australian context to improve nursing practice, education and research.

Paramedic knowledge of infectious disease aetiology and transmission in an Australian Emergency Medical System

Introduction: Paramedics and other emergency health workers are exposed to infectious disease particularly when undertaking exposure-prone procedures as a component of their everyday practice. This study examined paramedic knowledge of infectious disease aetiology and transmission in the pre-hospital care environment.--------- Methods: A mail survey of paramedics from an Australian ambulance service (n=2274) was conducted.--------- Results: With a response rate of 55.3% (1258/2274), the study demonstrated that paramedic knowledge of infectious disease aetiology and modes of transmission was poor. Of the 25 infectious diseases included in the survey, only three aetiological agents were correctly identified by at least 80% of respondents. The most accurate responses for aetiology of individual infectious diseases were for HIV/AIDS (91.4%), influenza (87.4%), and hepatitis B (85.7%). Poorest results were observed for pertussis, infectious mononucleosis, leprosy, dengue fever, Japanese B encephalitis and vancomycin resistant enterococcus (VRE), all with less than half the sample providing a correct response. Modes of transmission of significant infectious diseases were also assessed. Most accurate responses were found for HIV/AIDS (85.8%), salmonella (81.9%) and influenza (80.1%). Poorest results were observed for infectious mononucleosis, diphtheria, shigella, Japanese B encephalitis, vancomycin resistant enterococcus, meningococcal meningitis, rubella and infectious mononucleosis, with less than a third of the sample providing a correct response.--------- Conclusions: Results suggest that knowledge of aetiology and transmission of infectious disease is generally poor amongst paramedics. A comprehensive in-service education infection control programs for paramedics with emphasis on infectious disease aetiology and transmission is recommended.

Immunogenicity and protective efficacy of an anti-Streptococcus pyogenes vaccine candidate in multiple animal species

Streptococcus pyogenes, also known as Group A Streptococcus (GAS) has been associated with a range of diseases from the mild pharyngitis and pyoderma to more severe invasive infections such as streptococcal toxic shock. GAS also causes a number of non-suppurative post-infectious diseases such as rheumatic fever, rheumatic heart disease and glomerulonephritis. The large extent of GAS disease burden necessitates the need for a prophylactic vaccine that could target the diverse GAS emm types circulating globally. Anti-GAS vaccine strategies have focused primarily on the GAS M-protein, an extracellular virulence factor anchored to GAS cell wall. As opposed to the hypervariable N-terminal region, the C-terminal portion of the protein is highly conserved among different GAS emm types and is the focus of a leading GAS vaccine candidate, J8-DT/alum. The vaccine candidate J8-DT/alum was shown to be immunogenic in mice, rabbits and the non-human primates, hamadryas baboons. Similar responses to J8-DT/alum were observed after subcutaneous and intramuscular immunization with J8-DT/alum, in mice and in rabbits. Further assessment of parameters that may influence the immunogenicity of J8-DT demonstrated that the immune responses were identical in male and female mice and the use of alum as an adjuvant in the vaccine formulation significantly increased its immunogenicity, resulting in a long-lived serum IgG response. Contrary to the previous findings, the data in this thesis indicates that a primary immunization with J8-DT/alum (50ƒÊg) followed by a single boost is sufficient to generate a robust immune response in mice. As expected, the IgG response to J8- DT/alum was a Th2 type response consisting predominantly of the isotype IgG1 accompanied by lower levels of IgG2a. Intramuscular vaccination of rabbits with J8-DT/alum demonstrated that an increase in the dose of J8-DT/alum up to 500ƒÊg does not have an impact on the serum IgG titers achieved. Similar to the immune response in mice, immunization with J8-DT/alum in baboons also established that a 60ƒÊg dose compared to either 30ƒÊg or 120ƒÊg was sufficient to generate a robust immune response. Interestingly, mucosal infection of naive baboons with a M1 GAS strain did not induce a J8-specific serum IgG response. As J8-DT/alum mediated protection has been previously reported to be due to the J8- specific antibody formed, the efficacy of J8-DT antibodies was determined in vitro and in vivo. In vitro opsonization and in vivo passive transfer confirmed the protective potential of J8-DT antibodies. A reduction in the bacterial burden after challenge with a bioluminescent M49 GAS strain in mice that were passively administered J8-DT IgG established that protection due to J8-DT was mediated by antibodies. The GAS burden in infected mice was monitored using bioluminescent imaging in addition to traditional CFU assays. Bioluminescent GAS strains including the ‘rheumatogenic’ M1 GAS could not be generated due to limitations with transformation of GAS, however, a M49 GAS strain was utilized during BLI. The M49 serotype is traditionally a ‘nephritogenic’ serotype associated with post-streptococcal glomerulonephritis. Anti- J8-DT antibodies now have been shown to be protective against multiple GAS strains such as M49 and M1. This study evaluated the immunogenicity of J8-DT/alum in different species of experimental animals in preparation for phase I human clinical trials and provided the ground work for the development of a rapid non-invasive assay for evaluation of vaccine candidates.

The new middle class meets the creative class : the Master of Business Administration (MBA) and creative innovation in 21st-century China

‘MBA fever’ in China needs to be understood in the wider context of forces driving structural change in China’s relation to the global knowledge economy. The rise of a ‘new middle class’ in China is connected to the new claims for cultural leadership of an emergent ‘creative class’, which generates new issues about the relevance of the MBA in China, in terms of its relevance to Chinese economic circumstances, and its flexibility and capacity to respond to accumulation strategies that emphasise innovation, creativity and entrepreneurship.