Department of Ecology and Water Resources / MENVM

Department of Ecology and Resource Management / MENVSC

L'utilisation du pentobarbital de sodium (PB), injecté par voie intrapéritonéale (IP), est décrite comme une technique acceptable par les directives d'euthanasie de l'AVMA et du CCPA pour tuer les rongeurs. Cependant, de plus en plus de preuves contestent l'acceptabilité de l'IP PB. Celle-ci a été décrite comme inconsistante et il existe des données suggérant que cette technique pourrait induire de la douleur et du stress. L'objectif de cette thèse était donc de développer et d'évaluer des méthodes alternatives d'euthanasie. Au cours de l'étude pilote, nous avons développé un protocole d'injection pour les injections intrahépatiques (IH) de PB. Ensuite, nous avons testé cette injection sur des souris et des rats. Comme objectif secondaire, nous avons évalué l'utilisation de l'éthanol (ET) comme alternative au PB pour l’euthanasie des souris. Pour les souris, quatre-vingts souris CD1 adultes (mâles et femelles- 26,8 g [23-34 g], moyenne [intervalle]) ont été assignées au hasard à 6 groupes de traitement et ont été tuées par des injections IH ou des injections IP, en utilisant soit ET ou PB. Le taux de mauvaise injection (mauvais placement du contenu de l'injection) pour les essais IH était de 93% (28/30), y compris 14% intrathoracique (4/28), le reste ayant abouti dans la cavité péritonéale telle une injection IP. Ainsi, seulement 7% (2/30) des injections ont donné lieu à une administration hépatique (selon l’évaluation d'autopsie). Les injections IH ayant abouti dans le foie ont entraîné des décès quasi instantanés. Ces données montrent que les injections IH ne sont pas réalisables chez la souris étant donné la difficulté à frapper le foie et le risque d'injections intrathoraciques. D'autre part, l'IP ET a produit des temps significativement (p = 0.010; Mann-Whitney) plus courts de l'injection à l'arrêt du rythme cardiaque (CHB) (115s [88-185] médian [intervalle]) par rapport à l'IP PB (176s [123-260]), confirmant que l'ET est une alternative viable et potentiellement supérieure à la PB. Pour les rats, 66 injections IH et 14 injections IP ont été tentées sur des rats Sprague-Dawley mâles et femelles adultes (poids médian 371g, plage 170-730g), et ont entraîné un délai significativement plus rapide pour la perte du réflexe de redressement (LORR) (p < 0.0001, 95%CI 68 to 88s, Mann-Whitney) et temps de CHB (p < 0.0001, 95%CI 82 to 234s, Mann-Whitney) par rapport aux injections IP. Le temps médian de LORR et CHB après les injections IH était de 4s [1 to 96] et 142.5s [2 to 330] respectivement; alors que le temps médian de LORR et CHB après les injections IP était de 89.5s [73 to 110] et 275s [237 to 423], respectivement. Le taux de mauvaise injection, basé sur les évaluations d’autopsie, était plus élevé avec les injections IH qu'avec les injections IP (IH: 59%, IP: 29%); cependant, 97% des mauvaises injections IH ont tout de même produit une euthanasie réussie et rapide (LORR: 29s [1 to 96], CHB: 216s [12 to 330]. Les injections IH sont donc une alternative efficace aux injections IP pour l'euthanasie chez le rat, et présentent moins de risques d'échec des tentatives d'euthanasie. / The use of sodium pentobarbital (PB), injected intraperitoneally (IP), for killing rodents is described as an acceptable technique by the American Veterinary Medical Association (AVMA) and Canadian Council on Animal Care (CCAC) euthanasia guidelines. However, there is a growing body of evidence challenging the acceptability of IP PB. It has been described as inconsistent and there is evidence that it may induce pain and stress. The objective of this thesis was to develop and evaluate alternative methods of euthanasia. During the pilot study, an injection protocol for intrahepatic (IH) injections of PB was developed and then tested on both mice and rats. As a secondary objective, the use of ethanol (ET) was evaluated as an alternative to PB for mice. For mice, eighty adult (male and female) CD1 mice (26.8g [23-34g], mean [range]) were randomly assigned to 6 treatment groups and were killed by IH injections or IP injections, using either ET or PB. the misinjection rate (misplacement of injectate) for IH injections was 93% (28/30), including 14% intrathoracic (4/28), and the remainder were IP delivery. Only 7% (2/30) of IH attempts resulted in successful IH delivery, per necropsy evaluation. These yielded quasi-instantaneous deaths. These data show that IH injections are not feasible in mice given the difficulty in hitting the liver and the risk of intrathoracic injections. On the other hand, IP ET produced significantly (p = 0.010; Mann-Whitney) shorter time from injection to cessation of heartbeat (CHB) (115s [88-185] median [range]) compared with IP PB (176s [123-260]), confirming that ET is a viable and potentially superior alternative to PB. For rats, 66 IH injections and 14 IP injections were attempted on adult male and female Sprague-Dawley rats (median weight 371g, range 170-730g), and resulted in significantly faster time to loss of righting reflex (LORR) (p < 0.0001, 95%CI 68 to 88s, Mann-Whitney) and time to CHB (p < 0.0001, 95%CI 82 to 234s, Mann-Whitney) compared with IP injections. Time to LORR and CHB following IH injections were: LORR of 4s [1 to 96], CHB of 142.5s [2 to 330]; compared with IP injections: LORR of 89.5s [73 to 110], CHB of 275s [237 to 423). The misinjection rate was higher with IH injections than with IP injections (IH: 59%, IP: 29%); however, 97 % of IH misinjections resulted in fast and successful euthanasia (LORR: 29s [1 to 96], CHB: 216s [12 to 330]. IH injections are thus an efficacious alternative to IP injections for rat euthanasia and pose less risk of failed euthanasia attempts.

The main aim of the current study was to determine the prevalence and diversity of potentially zoonotic bacterial genera in accurately identified indigenous rodents from South Africa. Bacterial prevalence and diversity were determined by PCR amplification and sequence analyses. Rodents were molecularly identified by amplification and sequence analysis of the mitochondrial cytochrome b gene region. Three species (Aethomys ineptus, Mastomys coucha and Otomys angoniensis) belonging to murid species complexes were identified. Furthermore, phylogenetic analyses revealed that both the proposed subspecies (R. dilectus dilectus and R. d. chakae) within the recently erected Rhabdomys dilectus occur in Hammanskraal and at the University of Pretoria Experimental farm, both in the Gauteng Province of South Africa. An overall bacterial prevalence of 38.6 % was observed in kidney samples of commensal and natural indigenous rodents after molecular screening with broad range 16S rRNA gene primers. Nucleotide sequence analyses identified a diverse range of bacterial genera namely, Bartonella, Anaplasma, Helicobacter, Burkholderia, Streptococcus, Aerococcus and Lactobacillus. Some members of these genera have been identified as causative agents of human and animal diseases, being transmitted either through environmental contamination or through haematophagous arthropod vectors. Subsequent genus-specific bacterial screening focussed on vector-borne genera identified in the commensal and natural rodent populations sampled. Bartonella prevalence and genetic diversity was compared between a natural and commensal population of the southern multimammate mouse (M. coucha) using two gene regions (Citrate synthase gene and NADH dehydrogenase gamma subunit gene). A significantly higher infection prevalence was detected in the commensal population (92.9 %) as compared to the natural population (56.9 %). No differences however, were detected between infection status and the ectoparasite loads calculated for both rodent populations. Apart from several novel Bartonella strains identified in both M. coucha populations, phylogenetic analyses also identified a species of known zoonotic potential (B. elizabethae) in both populations. The present study represents one of the first to screen indigenous rodents for tick-borne members of the bacterial family Anaplasmataceae. Anaplasma bovis-like DNA was detected in five of the six rodent species sampled (A. ineptus, Lemniscomys rosalia, M. coucha, O. angoniensis and R. dilectus) at an overall prevalence of 39.2 %. The potentially zoonotic Ehrlichia ewingii was detected in M. coucha samples only at a prevalence of 5.3 %. The diverse bacterial genera detected in commensal and natural populations of indigenous rodents comprise members of zoonotic potential and agricultural significance, highlighting the importance of continuous disease surveillance of indigenous rodents. / Dissertation (MSc)--University of Pretoria, 2014. / National Research Foundation (NRF) / Zoology and Entomology / MSc / Unrestricted

Uppsatsen ämnar belysa kommensala gnagare inom arkeologin och förmedla potentialen för vidare forskning. Den medeltida bebyggelsen i Västergarn, Gotland agerar fallstudie där benen från små gnagare analyserats och diskuterats utifrån mikro- och makroarkeologins teoretiska vinklar. För den osteologiska analysen undersöktes benen med mikroskop för att se till ålder, spår av sjukdom och minsta individantal. I en rumslig analys sattes benen i jämförelse med de husgrunder som påträffats på platsen. Resultatet visade att materialet som fanns tillhandahållet endast innehöll de större benelementen, mest troligt till följd av den alltför stormaskiga sållen som användes vid utgrävningen. Benen som analyserades härrörde från mestadels mycket unga individer som befann sig i eller i anslutning till husgrunderna. Ett ben hade även en sjuklig förändring. Materialet visar på stor potential för framtida tvärvetenskaplig forskning där eventuella handelsrutter, sjukdomsrisker och matkonsumtion kan belysas. / This thesis aims to shed light on commensal rodents in archaeology and convey the potential for future research. The medieval settlement in Västergarn, Gotland was chosen for a case study where the rodent bones were analyzed and discussed based on micro- and macroarchaeological theoretical views. For the osteological analysis the bones were examined through a microscope to determine age, signs of disease and minimum number of individuals. The bones were put into context through a spatial analysis with the building foundations of the archaeological site. The results showed that the material at hand only contained the larger bone elements from the bodies, possibly because of the smaller bones falling through the big mesh sieve. The individuals were mostly very young and encountered near or within the building foundations. One bone had a pathological alteration. The material showed great promises for future interdisciplinary research where potential trading routes, risks of diseases and food consumption can all be made clearer.

In diese Dissertation, Ich konzentriere mich insbesondere darauf, wie die Artbestimmung in der Gattung Eimeria mit der Wirtsspezifität bei Nagetierarten zusammenhängt. Zunächst bietet diese Arbeit eine Reihe von Methoden zur Beurteilung der Prävalenz auf der Ebene der Parasitenarten in Mus musculus. Als Ergebnis war es möglich, drei verschiedene Eimeria-Spezies zu identifizieren, Mäuse mit Doppelinfektionen zu erkennen und die artenspezifische Prävalenz in Abhängigkeit von der Wirtsdichte vorherzusagen. Zur Identifizierung von Eimeria spp. über verschiedene Wirtsarten hinweg wurde eine neuartige Hochdurchsatz-Multi-Locus-Genotypisierungsmethode etabliert und mit der auf zuvor etablierten Markern basierenden Einzelmarker-Genotypisierung verglichen. Dies bestätigte, dass die Art E. falciformis in einer einzigen Wirtsart, der Hausmaus, vorkommt. E. vermiformis und E. apionodes konnten jedoch nicht unterschieden werden, was auf eine einzige Art mit breitem Wirtsspektrum hindeutet. E. vermiformis und E. apionodes konnten jedoch nicht unterschieden werden, was auf eine einzige Art mit breiter Wirtsverwendung in einem phylogenetischen Artkonzept schließen lässt. Diese Ergebnisse zeigen, dass die hohe Wirtsspezifität, die traditionell für Eimeria-Parasiten angenommen wird, fragwürdig ist und dass die Identifizierung von Arten durch Wirtsassoziation vermieden werden sollte. Durch molekulare Amplifikation, Sequenzierung, Genotypisierung und phylogenetische Analyse war es möglich, Eimerien auf Artniveau zu identifizieren und die Wirtsspezifität in Isolaten aus natürlichen Systemen in Frage zu stellen. In einer breiteren Perspektive betonte diese Arbeit die Notwendigkeit, Strategien bei der Erkennung, Quantifizierung und Identifizierung von Parasiten zu standardisieren und zu kombinieren, um ein besseres Verständnis auf evolutionärer und ökologischer Ebene zu erlangen. / This PhD thesis combines different approaches for parasite identification to assess the diversity of parasites in natural systems. Particularly, I focus on how species identification in the genus Eimeria is linked to its host specificity in rodent species. First, this thesis provides a set of methods to assess prevalence at the species level in Mus musculus systems. The approach integrates morphological description with molecular methods for detection, niche approximation and phylogenetic reconstruction. As a result, three different Eimeria species were identified, mice with double infections were detected and species-specific prevalence were predicted to be host density-dependent. For identification of Eimeria spp. across different host species, a novel high-throughput multi-locus genotyping was established and compared with single-marker genotyping. The multi-locus genotyping approach provided a higher resolution to distinguish closely related Eimeria isolates. This confirmed the species E. falciformis to have a single host species, the house mice. However, E. vermiformis and E. apionodes could not be distinguished suggesting a single species with broader host usage in a phylogenetic species concept. These findings show that the high host specificity traditionally assumed for Eimeria parasites is questionable, and that identification of species by host association should be avoided. The approaches for identification of Eimeria spp. Developed here allowed differentiation of closely related isolates with indistinguishable morphology. Molecular amplification, sequencing, genotyping and phylogeny allowed the identification of Eimeria at species level and to question host specificity in isolates from natural systems. In a broader perspective, this work emphasised the necessity to standardise and combine strategies in parasite detection, quantification and identification to gain better understanding at an evolutionary and ecological level.

Der Bewegungsapparat der Sciuromorpha, einer monophyletische Gruppe von ca. 300 Arten, wurde verwendet um den Effekt der Lebensweise und der Körpermasse auf die Scapula- und Femurmorphologie zu untersuchen. Diese Nagetierklade weist eine breite Vielfalt an Lebensweisen (arboreal, fossoriell, aerial) als auch Körpermassen (drei Größenordnungen umfassend) auf. Die fossorielle Lebensweise hat sich höchstwahrscheinlich dreimal unabhängig von einem arborealen Vorfahren entwickelt. Mehr als die Hälfte der rezenten Arten wurden untersucht. Die Scapulae wurden fotografiert, während Computertomographie (CT) und Oberflächenlaserscans für die Femora verwendet wurden. Es wurden funktionsrelevante Merkmale analysiert, wie die effektive Länge der Skelettelemente, die Muskeleigenschaften soweit aus der Geometrie der Knochen ableitbar, sowie die Robustheit. Die CT-Scans wurden verwendet, um die Querschnitts- und Trabekeleigenschaften des Femurs zu analysieren. Die Gestalt wurde mittels geometrischer Morphometrie untersucht. Phylogenetic comparative methods wurden unter anderem verwendet, um den Einfluss der Phylogenie zu beurteilen als auch, ob sich die unabhängige Aneignung einer fossoriellen Lebensweise in der Evolution homoplastischer Morphologien widerspiegelt. Die Phylogenie spielte bei der Merkmalsevolution eine vernachlässigbare Rolle. Das Auftreten signifikanter Merkmalsunterschiede zwischen den Lebensweisen sowie allometrischer Anpassungen aufgrund Veränderungen in der Körpermasse hingen von dem jeweiligen Merkmal ab. Bei einigen Merkmalen unterschied sich der Einfluss der Körpermasse signifikant zwischen den einzelnen Lebensweisen, was aber nicht die Regel zu sein scheint. Die Evolution homoplastischer Morphologien war sehr unwahrscheinlich bei den fossoriellen Gruppen. Diese Ergebnisse deuten auf eine komplexe, aber adaptive Evolutionsgeschichte dieser Skelettelemente bei den Sciuromorpha hin. / The vertebrate locomotor apparatus of Sciuromorpha, a monophyletic group of ca. 300 species, was used to investigate the effect of lifestyle and body mass on the scapular and femoral morphology. This rodent clade displays a broad diversity of lifestyles (arboreal, fossorial, aerial) and body masses spanning three orders of magnitude. The fossorial lifestyle evolved most probably three times independently from an arboreal ancestor. More than half of the extant species were included. Scapulae were photographed and computed tomography (CT) and surface laser scans were acquired for the femora. Functionally relevant traits were analysed, e.g., the effective length of the skeletal element, the properties of attaching muscles as inferred from the geometry of the bones, and robustness properties. The CT scans were used to analyse the cross-sectional and trabecular properties of the femur. Bone shape was investigated using geometric morphometrics. Phylogenetic comparative methods were utilized, e.g., to assess phylogenetic inertia and whether the independent acquisition of a fossorial lifestyle is reflected in the evolution of homoplastic morphologies. Phylogenetic inertia played a neglectable role in the trait evolution. Significant differences among lifestyles as well as allometric scaling adjustments depended on the trait under consideration. For some traits, scaling differed significantly among lifestyles, although this did not appear to be the rule. Morphological homoplasy was unlikely among the fossorial groups. The results suggest a complex, but adaptive evolutionary history of these skeletal elements in Sciuromorpha.

Thesis (PhD)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: The emergence and re-emergence of viral human pathogens from wildlife sources in the recent past has led to increased studies and surveillance of wildlife for potentially zoonotic agents in order to gain a better understanding of the pathogens, their sources as well as events that may lead to viral emergence. Of the >1407 known human pathogens, 13% are classified as emerging or re-emerging, and 58% as zoonotic; 37% of the (re-)emerging and 19% of the zoonotic pathogens are RNA viruses, accounting for the majority of recently emerged infectious diseases with a zoonotic origin, such as HIV, Ebola, Hendra, Nipah, Influenza and SARS. This study focusses on potentially zoonotic viruses hosted by rodents (Muridae family), shrews (order previously known as Insectivora/Soricomorpha, now reclassified as Eulipotyphla) and bats (order Chiroptera). Rodents and bats represent the largest (~40%) and second largest (~25%) mammalian orders and both occur on every continent except Antarctica. Together, the three mammalian orders investigated represent the most relevant potential sources of new zoonoses. In this study I investigated the occurrence of astroviruses, arenaviruses, coronaviruses and hantaviruses in South African small mammal species belonging to the orders mentioned above. These viruses have either been implicated in recent emerging zoonotic events or are considered to have the potential to cause cross-species transmissions resulting in a zoonotic event. In the first part of the study specimens collected from various bat, rodent and shrew species were screened for viral sequences by broadly reactive PCRs; positive samples were characterised by sequencing and sequence analysis. A separate part of the study focussed on hantavirus disease in humans: a seroprevalance survey was conducted to determine the presence of hantavirus antibodies in the local population. Additionally, acutely ill patients with potential hantavirus disease were tested in an attempt to identify possible acute infections and define clinical hantavirus disease in South Africa. Screening of rodent and shrew specimens resulted in the identification of eight novel arenavirus sequences. Seven of the sequences are related to Merino Walk virus, a recently identified South African arenavirus, and the eighth sequence represents a novel lineage of Old World arenaviruses. Screening of bat specimens resulted in the identification of highly diverse novel astrovirus and coronavirus sequences in various South African bat species, including the identification of a viral sequence closely related to the recently emerged Middle East Respiratory Syndrome coronavirus. While the study did not identify hantavirus infections in any of the acutely ill patients, it found seroprevalences similar to those observed in Europe and West Africa. The results obtained highlight the importance of small mammals in the emergence of potential zoonoses and further reinforce the importance of viral surveillance of relevant wildlife species. Further in-depth studies of naturally infected reservoir host populations are required in order to gain a better understanding of virus-host dynamics and the events that lead to virus emergence. / German Research Foundation (DFG) (project number: KR1293/9-1/13-1) / The Polio Research Foundation and the NHLS Research / Harry Crossley Foundation, the Polio Research Foundation and Stellenbosch University for granting scholarships and bursaries for PhD.

The ability to perceive the Earth's magnetic field has been demonstrated in a variety of animals, including representatives of all five classes of vertebrates. The physiological mechanisms underlying magnetic field sensation, however, remain largely unknown. Behavioral, physiological, neuroethological studies and studies using early response genes as neuronal activation markers indicated that a major role in the perception and processing of magnetic information play trigeminal, vestibular and visual systems. Subsequently, magnetic information seem to be integrated with multimodal sensory and motor information within the hippocampal-entorhinal system. In the majority of studies, however, birds have been used as model organisms. In this work I analyzed the neural substrate of magnetic compass orientation in the mouse strain C57BL/6J using markers c-Fos and Egr1. I found that all the aforementioned systems contain neurons responsive to the experimental magnetic fields. This finding demonstrates a complex processing of the magnetic information at level of the central nervous system.

Dentre os ecossistemas neotropicais, a Mata Atlântica é considerada um dos mais importantes hotspots mundiais. O presente estudo foi conduzido na Reserva Florestal Morro Grande - RFMG (23°39\'-23°48\'S, 47°01\'-46°55\'W), reconhecida pelo seu alto valor para a conservação e está localizada na faixa da Mata Atlântica Ombrófila Densa Montana, Planalto Atlântico do Estado de São Paulo. Presentes na Mata Atlântica, os pequenos mamíferos não-voadores constituem o grupo de mamíferos mais diverso do bioma, e dados recentes relacionados à representatividade ecológica sugerem que os Orizomíneos mais típicos, comuns e abundantes das florestas costeiras e de planalto no estado de São Paulo são Euryoryzomys russatus e Sooretamys angouya. Estes dados ainda indicam que E. russatus e S. angouya, espécies classificadas respectivamente como \"em risco de extinção\" e \"deficiente de dados\" no Estado de São Paulo, respondem diferentemente ao processo de fragmentação, mas não existem informações suficientes disponíveis de história natural e autoecologia para o melhor entendimento destas respostas ao ambiente. O presente projeto avaliou a estrutura populacional, a área de vida, o uso do espaço, a dieta e seleção alimentar exibida por E. russatus e S. angouya na RFMG. O presente trabalho demonstrou que a espécie E. russatus apresenta uma abundância maior do que S. angouya na RFMG e que a temperatura e a disponibilidade de frutos influenciam a variação populacional de E. russatus, enquanto que, para S. angouya, a variação populacional independe dos fatores bióticos (frutos e artrópodes) ou abióticos (temperatura e precipitação) avaliados. Foi verificado uma estratificação vertical no uso do espaço para S. angouya e E. russatus, já que S. angouya apresentou uma locomoção escansorial enquanto E. russatus se apresentou estritamente terrestre. O estudo sugere que a disponibilidade de recursos, o período reprodutivo e o tamanho do indivíduo são os principais fatores que afetam o tamanho de área de vida, o uso do espaço e a locomoção apresentada pelas espécies. Os resultados obtidos pelo estudo da dieta, demonstram de forma conclusiva que E. russatus seleciona alimentos de origem animal, e que S. angouya utiliza muito pouco, ou não utiliza, fontes de origem animal, mas sim fontes vegetais ricas em proteínas e carboidratos, como os frutos. Este estudo possibilitou a compreensão de estratégias de vida adotadas por E. russatus e S. angouya, o que permitiu uma análise comparada da história natural a partir de um contexto evolutivo de organismos que compartilham a mesma escala geográfica e temporal, o que é algo inédito dentro da tribo e da subfamília. / The Atlantic Forest is considered one of the most important global hotspot among the neotropical ecosystems. This study was conducted at the Morro Grande Forest Reserve - MGFR (23°39\'-23°48\'S, 47°01\'-46°55\'W), located in the Dense Montana Atlantic forest, Atlantic Plateau of São Paulo, Brazil, which is known by its high conservation value. The non-flying small mammals are the most diverse group of mammals in the Atlantic forest and recent data suggest Euryoryzomys russatus and Sooretamys angouya as the most common and abundant species in coastal forests and highlands in the state of São Paulo. These data also indicate that E. russatus is classified as endangered and S. angouya as data deficient in the state of São Paulo red list, and these species respond differently to the fragmentation process. This project evaluated the population structure, the living area, the use of space, the diet, and food selection displayed by E. russatus and S. angouya in MGFR. This study demonstrated that the species E. russatus features greater abundance than S. angouya in the study area. Thus, the temperature and the availability of fruits influenced the variation of the population of E. russatus, whereas the population variation of S. angouya showed no dependence of assessed biotic (fruits and arthropods) or abiotic factors (temperature and precipitation). Vertical stratification was observed in the use of space between S. angouya and E. russatus, once S. angouya presented escansorial locomotion and E. russatus was strictly terrestrial. The study suggests that the availability of resources, the reproductive period and the individual overall size are the main factors that affect the home range size, the use of space and mobility presented by the species. The results obtained by the study of diet demonstrate conclusively that E. russatus selects animal origin and S. angouya uses very little or does not use animal food resources, choosing plant sources rich in protein and carbohydrates, such as fruits. This study brought a better understanding of the life strategies adopted by E. russatus and S. angouya, which allowed the comparison of natural history of organisms that share the same spatial and temporal scale under an evolutionary perspective, which is a completely new approach within the tribe and subfamily.