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Haemorrhagic shock (HS) is commonly associated with trauma. Severe haemorrhage causes hypoperfusion of tissues resulting in a global ischaemic state, and resuscitation is performed to restore circulating volume. However, the return of oxygen to ischaemic tissues causes the induction of a systemic inflammatory response, which contributes to cell death leading to organ failure. In trauma patients, failure of more than four organs is linked to certain mortality, highlighting the need for interventions that may reduce or prevent the deterioration in organ function. The aim of this thesis was to investigate the effect of therapeutic approaches on the organ injury and dysfunction induced by HS. Briefly, male Wistar rats were subjected to haemorrhage by withdrawal of blood to reduce the mean arterial pressure to 35 ± 5 mmHg for 90 min. Followed by resuscitation with 20 ml/kg Ringer’s lactate for 10 min and 50% of the shed blood for 50 min. Organ function was determined 4 h after the onset of resuscitation. This model was used to investigate the effect of three different interventions on the organ injury and dysfunction induced. In the first study, administration of bone marrow-derived mononuclear cells (BMMNCs) upon resuscitation resulted in (1) significant attenuation of the organ injury and dysfunction associated with HS, and (2) restoration of the activation of the Akt pro-survival pathway. It is possible that these beneficial effects are mediated by paracrine mediators secreted by BMMNCs, which modulate this pathway, however injection of large numbers of cells is not practical in the acute setting of trauma. Therefore, in the next study erythropoietin (EPO) was used as a daily pre-treatment for three days prior to the induction of haemorrhage, as EPO is a known stimulus of endothelial progenitor cell (EPC) mobilisation. EPO pre-treatment resulted in (1) significant attenuation of the organ injury and dysfunction associated with HS, (2) mobilisation of EPCs (CD34+/flk-1+), and (3) activation of the Akt pro-survival pathway with enhanced activation of eNOS. However, when used clinically EPO is associated with an increased risk of thrombotic events, therefore in the final study a non-erythropoietic analogue of EPO was investigated. Treatment with pyroglutamate helix B surface peptide (pHBSP) resulted in (1) significant attenuation of the organ injury and dysfunction associated with HS, and (2) activation of the Akt pro-survival pathway with enhanced activation of both eNOS and STAT3. Additionally, late pHBSP treatment, up to 60 min after the onset of resuscitation, exerted the highest degree of protection. The findings of this thesis support the view that modulation of the Akt pro-survival pathway is a potential therapeutic target in the treatment of the ischaemia-reperfusion injury associated with severe haemorrhage and resuscitation.

The project was concerned with the construction of defined attenuated derivatives of P. multocida serotype B:2 strains, causative agents of haemorrhagic septicaemia, and attempts were made to construct defined mutations in genes such as aroA, cya, and galE loci that have been used to induce attenuation in other bacterial strains. Mutants defective in the aroA gene were constructed by allelic exchange of the locus in the chromosome of the wild-type strains with a cloned aroA gene interrupted with a cassette encoding kanamycin resistance (KmR). The aroA defective strains were confirmed by PCR, Southern blotting, lack of growth on minimal medium and by enzyme assay. KmR inactivated aroA mutants JRMT1 and JRMT2 strains derived from P. multocida 85020 and Quetta strains, respectively, were highly attenuated in a mouse model, with an LD50 108 C.F.U./mouse after injection intraperitoneally (i.p.). In contrast, the wild-type strains had LD50 <50 C.F.U./mouse by this route. Vaccination once by the i.p. route or twice by the i.n. route with these aroA mutants gave complete protection to the mice against subsequent challenge i.p. with 10,000 LD50 of the homologous wild-type strain or 1000 LD50 of the heterologous wild-type strain. Vaccination with these by the s.c. route was not protective. When high doses of the attenuated strains were inoculated by the i.p. or i.n. routes, there was some spread to the internal organs but the organisms were cleared by 24 and 72 hrs respectively. In contrast, the wild-type parent strains spread rapidly and multiplied in high numbers and killed the mice by 24 and 96 hrs respectively.

MSc (Med) (Bioethics and Health Law), Faculty of Health Sciences, University of the Witwatersrand

Rift Valley fever (RVF) belongs to the group of viral haemorrhagic fevers (VHFs), most of which are zoonotic diseases causing outbreaks in animals and humans all over Africa. In the absence of haemorrhagic or specific organ manifestations, these diseases are clinically difficult to diagnose. Rapid laboratory confirmation of cases is therefore essential for timely execution of supportive treatment, appropriate case management, infection control, and tracing of contacts. Rift Valley fever virus (RVFV), a mosquito-borne pathogen, is responsible for high mortality rates and abortion in domestic ruminants, resulting in significant socio-economic losses. Furthermore, the virus is potentially infectious by aerosol, can replicate in a wide range of mosquito species and poses a bioweapon threat. The recent spread of the virus outside of the African continent, demonstrates its ability to move northwards to RVF free regions, e.g. to Europe and Northern America. Such fears fuel the international demand for reliable and validated diagnostic tools for rapid diagnosis of RVF. The aim of this study was to develop a rapid and accurate molecular tool for the detection of RVFV. A real-time loop-mediated isothermal amplification assay (LAMP) targeting the L segment of RVFV, was developed and evaluated. The assay proved to be highly specific and able to detect RVFV strains representing the genetic spectrum of the virus. Furthermore, the assay did not amplify the RNA of other genetically and antigenically related phleboviruses. The sensitivity of the assay was compared to that of a previously published TaqMan RTD-PCR protocol and found to be equal. Similarly, the assay demonstrated very high diagnostic sensitivity and specificity in various clinical human and animal specimens, collected during natural outbreaks of the disease in Africa. The detection of specific viral genome targets in positive clinical specimens was achieved in less than 30 minutes. As a highly accurate, rapid and very simple nucleic acid detection format, the RT-LAMP assay has the potential to be used in less well equipped laboratories in Africa. The assay format can be adapted to a portable device that can be utilized during RVF outbreaks in remote areas, and can be a valuable tool for differential diagnosis of VHFs. / Dissertation (MSc)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

In the past five years generally well managed farms reported an increase in acute deaths in their grower herds to their consulting veterinarian. At the same time reports from across the world indicated that this is not a problem seen only in South Africa. The syndrome is generally referred to as haemorrhagic bowel syndrome (HBS), red gut or balloon pig. Veterinarians generally believed that the cause of these acute deaths were due to the acute form of Lawsonia intracellularis, also known as porcine haemorrhagic enteropathy (PHE). Because neither the clinical symptoms present prior to death, nor the post mortem changes were typical for a L. intracellularis case it was decided to investigate this syndrome in more depth. Five commercial farms were purposefully selected where growers that died peracutely were necropsied and intestinal samples collected for histological as well as bacteriological examination. A total of 28 pigs were sampled with the histological sections from all samples indicating a Clostridium species as the cause and from 11 of samples Clostridium perfringens were cultured as the predominant bacterium. Although pigs on the farms were seropositive for Lawsonia intracellularis there was no evidence that this bacterium was the cause of death in the pigs. Rather the aetiology points to C. perfringens being the cause, possibly together with other predisposing factors such as rapid growth, high ambient temperatures and interruption in fedding patterns. Based on these results further studies to determine the toxin type as well as predisposing factors should be done. Copyright / Gedurende die afgelope vyf jaar het plase met ’n algemene goeie bestuur ’n verhoging in akute vrektes in hulle groeikuddes opgemerk en hulle het hulle kommer oor die vrektes aan hulle konsulterende veeartse oorgedra. Diè verhoging in groeivrektes is nie uniek aan Suid Afrika nie. Dieselfde tendens is regoor die wêreld opgemerk, maar niemand is seker wat presies die oorsaak van die akute vrektes is nie. In die literatuur word daar na “haemorrhagic bowel syndrome (HBS)” oftewel hemoragiese derm sindroom verwys. Boere verwys na die sindroom as rooiderm of “balloon pig”. Tot nou toe het veeartse aanvaar dat die oorsaak moontlik Lawsonia intracellularis is. Die organisme is verantwoordelik vir ’n groep sindrome waarvan “porcine haemorrhagic enteropathy” die akute form is. Omrede die kliniese simptome en die nadoodse ondersoek nie tipies vir ’n L. Intracellularis geval is nie, is daar besluit om die akute vrektes verder te ondersoek. Vyf plase, waar die sindroom baie voorkom, is geidentifiseer en dermonsters is geneem vir histopatalogiese sowel as mikrobiologiese ondersoeke. In totaal is monsters van 28 varke geneem. Die histologies seksies van al die monsters het gedui op ’n Clostridium spesie as die hoofoorsaak van vrekte en Clostridium perfringens is uit 11 van die monsters geisoleer. Alhoewel al 5 plase serologies positief getoets is vir Lawsonia intracellularis, was daar geen bewyse gewees dat die bakterium verantwoordelik vir die vrektes was nie. Die etiologie dui eerder op C. perfringens as die oorsaak. Daarby saam speel ander faktore soos vinnige groei, hoë omgewingstemperature asook onderbrekings in beskikbaarheid van voer heelwaarskynlkik ’n belangrike rol in die sindroom. Verdere navorsing om die toksien tipe te identifiseer asook die identifikasie van moontlike faktore wat die sindroom aanhelp moet gedoen word. / Dissertation (MMEdVet)--University of Pretoria, 2009. / Production Animal Studies / unrestricted

Canine parvovirus type 2 (CPV-2), the aetiological agent of haemorrhagic enteritis in dogs emerged in 1978 worldwide. In the mid 1980’s, the original CPV-2 had evolved and was completely replaced by 2 variants, CPV 2a and 2b. In 2000, a new variant of CPV (CPV-2c) was detected in Italy and now circulates in other countries. Haemorrhagic enteritis in dogs is a major disease in South Africa and Nigeria. Both infection rates with CPV-2 and case fatality rates in young dogs are high. CPV-2 is a small, negative-sense, single-stranded DNA virus of 5.2kb long and a member of the Parvoviridae family, which also includes feline panleukopenia virus (FPV) and mink enteritis virus (MEV). The CPV-2 genome is prone to mutations at the VP2-encoding region. As a result we investigated the genetic composition of the VP2 region in the CPV-2 genome using molecular methods (qPCR) to provide information for comparison of field and vaccine strains of the virus. The conventional PCR detection results yielded 137 (97.85%) of the total of 140 feacal samples screened with diarrhoea positive. One hundred-and-six of 108 samples from South Africa (98.15%) tested positive and two (1.85%) were negative, while 30 (96.77%) from 31 faecal samples from Nigeria were positive and 1 (2.23%) was negative. Results obtained from the genotyping of the CPV- 2 strains using CPV-2a/b and CPV-2b/c TaqMan assays employing minor groove binder (MGB) probes, revealed that out of a total of 106 South African samples, 100 (94.34%) were infected with CPV-2b and 6 (5.66%) with CPV-2a, while all the Nigerian samples [n=30 (100%)] contained only CPV2a. There was no reported case of CPV-2c. The VP2 gene of selected DNA samples (n=27), from South Africa (n=19), Nigeria (n=6) and multivalent vaccines (n=2) were amplified and sequenced. These sequences were originally aligned and edited to a total length of 1,750 bp of the CPV-2 VP2 encoding gene. These selected sequences showed 99% maximum identity to the GenBank sequences from the blast results (NCBI BLASThttp:// www.ncbi.nlm.nih.gov/BLAST/) and alignment of all the sequences was performed using ClustalX. Two phylogenetic analyses showed most South African field isolates distant from viruses from other parts of the world. A few clustered with Asian and European strains, while Nigerian CPV-2 strains clustered with USA and some European isolates. The results of the protein analysis showed seven changes of amino acids at positions 265, 297, 324, 424, 426, 440 and 475 for most of the South Africans strains while the Nigerian CPV-2 had only one field isolate with an amino acid change. / Dissertation (MSc)--University of Pretoria, 2010. / Veterinary Tropical Diseases / unrestricted

INTRODUÇÃO: A ressuscitação de baixo volume com solução salina hipertônica (SSH) ou terlipressina pode ser uma alternativa à administração de grandes volumes de cristaloides no tratamento do choque hemorrágico. O objetivo deste estudo foi avaliar os efeitos da HHS e terlipressina sobre a perfusão e oxigenação cerebral e investigar os mecanismos cerebrais envolvidos na microcirculação, função mitocondrial, atividade eletrocortical e vias apoptóticas cerebrais durante choque hemorrágico. MÉTODOS: Animais anestesiados com isofluorano foram submetidos ao choque hemorrágico [grupo Hemo; pressão arterial média (PAM) de 40 mmHg por 30 minutos] e tratados com Ringer lactato (RL) (3RL; 3x volume de sangue removido), terlipressina (grupo Terli; bolus) ou SSH (grupo SSH; 4 mL/kg bolus) e comparados ao grupo Sham. Um modelo porcino (n = 56) foi utilizado para avaliação da pressão de perfusão cerebral (PPC) e de oxigênio tecidual (PbtO2), e da expressão cerebral de marcadores teciduais da regulação de água (aquaporina-4), sódio (cotransportador-1 de Na-K-2Cl), estresse oxidativo (substâncias reativas ao ácido tiobarbitúrico e superóxido dismutase dependente de manganês) e apoptose. Um modelo murino (n = 179) foi utilizado para avaliação da microcirculação (fluorescência de FITC-dextrano) e função mitocondrial (potencial redox e de membrana mitocondrial, utilizando-se a fluorescência de flavoproteínas endógenas e do tetrametilrodamina metil éster, respectivamente) no córtex cerebral, utilizando-se a microscopia confocal in vivo, e para avaliação da atividade eletrocortical cerebral, por meio da monitorização do potencial evocado somatossensorial. No modelo murino foram avaliados três grupos adicionais, constituídos pela associação da terlipressina ao RL (1x, 2x ou 3x volume removido). RESULTADOS: No grupo Hemo porcino, houve uma redução significativa da PPC e PbtO2, associada ao aumento na expressão cerebral de marcadores da regulação do transporte de água e sódio, estresse oxidativo e apoptose em relação ao Sham. No modelo murino, a hipotensão induzida pelo choque hemorrágico foi correlacionada à diminuição na densidade vascular cortical e às disfunções mitocondriais e da atividade eletrocortical cerebral. No grupo 3RL porcino, a infusão de grandes volumes de RL recuperou a PbtO2, mas não a PPC, e foi acompanhada por uma maior expressão cerebral de marcadores da regulação de água, estresse oxidativo e apoptose comparada ao Sham. Nos ratos, a ressuscitação volêmica agressiva não recuperou a densidade vascular cortical, que foi correlacionada às disfunções mitocondrial e da atividade eletrocortical. No grupo Terli porcino, o aumento na PAM foi associado à restauração da PPC, PbtO2 e expressão dos marcadores da regulação de água e sódio, estresse oxidativo e apoptose no cérebro. Nos ratos tratados com terlipressina, associada ou não a 1x ou 2x RL, houve uma correlação positiva entre a recuperação da densidade vascular cortical e a restauração das funções mitocondrial e atividade eletrocortical cerebral. A SSH não promoveu melhora em nenhum dos modelos. CONCLUSÕES: RL e terlipressina recuperaram a oxigenação no córtex cerebral, mas apenas a terlipressina recuperou a perfusão cerebral, revertendo as disfunções mitocondrial e eletrocortical no cérebro e o aumento no transporte de água e sódio, estresse oxidativo e apoptose induzidos pelo choque hemorrágico. A SSH não recuperou a perfusão e oxigenação cerebral / INTRODUCTION: Small-volume resuscitation with hypertonic saline solution (HSS) or terlipressin can be an alternative to the administration of large amounts of crystalloids in haemorrhagic shock. The aim of this study was to evaluate the effects of HSS and terlipressin on cerebral perfusion and oxygenation and investigate the cerebral mechanisms associated with microcirculation, mitochondrial function, electrocortical activity and apoptotic pathways during haemorrhagic shock. METHODS: Isoflurane-anaesthetised animals were submitted to haemorrhagic shock [Haemo group; mean arterial pressure (MAP) of 40 mmHg for 30 minutes] and treated with lactated Ringer\'s solution (LR) (3LR group; 3x volume bled), terlipressin (Terli group; bolus) or HSS (HSS group; bolus 4 mL/kg) and were compared with a Sham group. A porcine model (n = 56) was used to assess the cerebral perfusion pressure (CPP) and tissue oxygenation (PbtO2) and the expression of tissue markers of water (aquaporin-4), sodium (Na-K-2Cl cotransporter-1), oxidative stress (thiobarbituric acid reactive substances and manganese superoxide dismutase) and apoptosis in cerebral samples. A murine model (n = 179) was used to assess microcirculation (FITC-dextran fluorescence) and mitochondrial function (redox and membrane potential, using the fluorescence of endogenous flavoproteins and tetramethylrhodamine methyl ester, respectively) in the cerebral cortex by using in vivo confocal microscopy, and to assess the electrocortical brain activity by monitoring the somatosensory evoked potential. In the murine model, three additional groups were evaluated, which received terlipressin associated to LR (1x, 2x or 3x blood withdrawn). RESULTS: In the porcine Hemo group, there was a significant decrease in the CPP and PbtO2, which were associated to an increased cerebral expression of markers of water and sodium transport, oxidative stress and apoptosis compared with Sham. In the murine model, the haemorrhagic shock-induced hypotension was correlated to a decrease in the cortical vascular density and to dysfunctions on brain mitochondria and electrocortical activity. In the porcine 3LR group, the infusion of large volumes of LR recovered the PbtO2, but not the CPP, and was accompanied by an increased cerebral expression of markers of water and sodium transport, oxidative stress and apoptosis compared with Sham. In the rats, the aggressive fluid resuscitation did not recover the cortical vascular density, which was correlated to the brain mitochondrial and electrocortical dysfunctions. In the porcine Terli group, the increase in the MAP was associated with the recovery of CPP, PbtO2, and expression of markers of water and sodium regulation, oxidative stress and apoptosis within the brain. In the rats treated with terlipressin, associated or not with 1x or 2x LR, there was a positive correlation between the recovery of the cortical vascular density and the recovery of the brain mitochondrial and electrocortical functions. Such improvements were not observed in none of the models treated with HSS. CONCLUSIONS: LR and terlipressin recovered tissue oxygenation in the cerebral cortex, but only terlipressin recovered the cerebral perfusion, reversing the brain mitochondrial and electrocortical dysfunctions and the increase in the markers of water and sodium transport, oxidative stress, and apoptosis induced by haemorrhagic shock. The HSS did not recover cerebral perfusion and oxygenation

Organisms are continuously exposed to heterogeneous micro- and macro-parasitic species, hence simultaneous infections often occur in wild and farm environments. This joint project aimed to develop a co-infection model between chronic and acute infections, evaluating their impact on the fish immune system. Proliferative Kidney Disease was studied on farmed rainbow and brown trout during natural seasonal outbreaks, using a parasite gene (Tetracapsuloides bryosalmonae RPL18) as a proxy for assessment of parasite burden. In hosts with elevated susceptibility PKD pathogenesis was shaped by an anti-inflammatory phenotype, a profound B cell/antibody response and dysregulated TH cell-like activity. Pathogen-free brown trout were exposed to Viral Haemorrhagic Septicaemia Virus (comparatively using European VHSV-Ia and North American VHSV-IVb strains) or to the bacterium Yersinia ruckeri. This European native species was highly resistant to the VHSV-IVb strain, which was undetectable in internal organs despite raising a strong antiviral and mucosal immune response. Following VHS and Yersiniosis infection, haemo-lymphopoietic organs were screened by RT-qPCR to assess the specific pathogen burdens and characterise the immune responses elicited. Transcription patterns were analysed for Interferons, CXC chemokines, SOCS (potential disease resistance biomarkers) and genes of the PACAP system. Lastly, PKD-infected brown trout were co-infected with VHSV-Ia, resulting in typical lesions while showing reduced and delayed mortality. PKD+/VHS+ fish were identified by RT-qPCR and histopathology screening. Pro-inflammatory and antimicrobial peptide genes were modulated following virus co-infection when compared to fish with single infection, with an earlier activation of cellular and humoral responses, and a stronger up-regulation of TH1 and antiviral genes. Oligonucleotide microarrays were used to assess the broader immune gene transcription modulation between single- and co-infected fish. Overall, the results suggest that the immune response of brown trout might be enhanced during the PKD/VHS co-infection.

Le virus Ebola, responsable d'une fièvre hémorragique virale létale et le virus de la variole, agent étiologique de la variole, sont des armes biologiques potentielles. Il n'existe pas de traitement ou de prophylaxie autorisés contre le virus Ebola, quelques candidats vaccins étant en cours de développement. Concernant la variole, des vaccins dits de première génération (virus de la vaccine) ont permis l'éradication de la maladie cependant ils sont à l'origine de complications post-vaccinales parfois sévères alors que des vaccins plus récents dits de troisième génération, non-réplicatifs, ont été développés pour leur innocuité mais restent faiblement immunogènes. Nous avons récemment développé plusieurs vecteurs viraux de type virus de la vaccine (VACV) par délétion d'un certain nombre de facteurs de virulence. Nous avons évalué leur innocuité, leur immunogénicité et leur efficacité en tant que candidats vaccins antivarioliques chez la souris puis utilisé l'un de ces vecteurs pour développer un candidat vaccin bivalent antivariolique et anti-virus Ebola. Ces virus de la vaccine délétés sont réplicatifs mais fortement atténués. Ils induisent une réponse en anticorps neutralisants spécifiques anti-vaccine similaire à celle induite par le vaccin antivariolique de première génération et induisent des réponses immunitaires cellulaires CD4+ et CD8+ spécifiques suffisantes pour protéger l'animal d'un challenge létal de cowpoxvirus en intranasal, simulant une infection par le virus de la variole. Le virus délété le plus immunogène et le plus sûr, nommé MVL, a été utilisé pour construire un vecteur viral codant pour la glycoprotéine du virus Ebola (EGP). Le gène entier d'EGP ou une forme chimérique d'EGP (fusion entre l'ectodomaine d'EGP et le domaine transmembranaire de la glycoprotéine B5 du VACV) ont été clonés dans le génome du vecteur viral. Ces deux vecteurs produisent des virus ayant incorporé EGP dans leur enveloppe. Ces deux candidats vaccins recombinants induisent de fortes réponses humorales spécifiques anti-EGP et anti-vaccine chez la souris immunocompétente. En conclusion, nous avons développé plusieurs candidats vaccins antivarioliques aussi immunogènes et efficaces que le vaccin historique et avec une atténuation similaire aux vaccins de troisième génération. L'un de ces candidats (MVL) a été utilisé comme vecteur viral pour exprimer la glycoprotéine hétérologue EGP, contre laquelle il induit une réponse immunitaire humorale forte / Ebola virus, causing a lethal haemorrhagic fever and variola virus, the agent of smallpox are potential biological weapons. There is no treatment and no prophylaxis authorised against Ebola, although some vaccine viral vectors were developed these last years. Concerning smallpox, several types of vaccines exist against smallpox (based on vaccinia virus), first generation that allowed the disease eradication but responsible of some post-vaccination complications and some non-replicative 3rd generation vaccines which are safe but not very immunogenic. We have recently developed several vaccinia virus (VACV) vectors by deletion of some virulence genes, and we have evaluated their safety, immunogenicity and efficacy as smallpox vaccine in mice and used one of them as a bivalent vaccine against Ebola and smallpox. These viral vectors are higly attenuated and replicative competent. They induce a neutralizing specific-VACV antibodies response similar to that of the historical vaccine and induce VACV-specific CD8+ and CD4+ immune responses efficient to protect immunocompetent mouse model intranasally infected by cowpox virus, simulating variola virus infection.The most safety and immunogenic vaccinia virus vector, named MVL, has been used to construct a vector encoding the Ebola glycoprotein (EGP) for immunization against Ebola. The native EGP gene or a chimeric EGP gene (a fusion between the EGP ectodomain and the transmembrane domain of the VACV B5 glycoprotein) have been cloned into the viral vector genome. These two recombinant vaccine candidates induce specific humoral immune responses against Ebola and vaccinia virus in immunocompetent mice. In conclusion, we have developed several vaccine candidates against smallpox as immunogenic and protective as the historical vaccine and as safe as 3rd generation vaccines. One of these candidates, MVL, has been used as a viral vector to express the heterologous glycoprotein EGP, against which it induce a strong humoral immune response.