Made available in DSpace on 2016-01-26T18:55:33Z (GMT). No. of bitstreams: 1 thais.pdf: 774762 bytes, checksum: aab7c8f9a2cbb52b94d59cd3cc962c07 (MD5) Previous issue date: 2011-10-24 / The sheep industry now occupies an important place of interest within the livestock farms, requiring the development of studies in the area related to sheep breeding on tropical climate. Most of the wool breed have reproductive seasonality, in other words, cycles are controlled by the fading light of the season, on the other hand crossbred sheep usually have already observed reproductive activity throughout the year. The ovarian cortex has follicles and corpus luteumin various stages of development, supported by connective tissue and smooth muscle fibers through the vessels and nerves. Te ovarian marrow is made up of fibroelastic tissue, nerves and arteries, being the innermost part of the ovary. In antral follicles, IGF stimulates the proliferation activity and differentiation of granulosa cells and steroidogenesis. The nucleolar organizer regions (NORs) are loops of deoxyribonucleic acid (DNA) responsible for synthesizing ribosomal ribonucleic acid (rRNA) during interphase, the period of protein synthesis and increase in volume, size and number of cell organelles. Its in the nucleolus, located in the cell nucleus, which has storage and production of rRNA, which will be linked to various proteins to form ribossomes. The AgNOR technique evaluates the NORs of active chromossomes during the cell cycle and the number or area of NORs show that proliferative activity, from the G1 phase of intense production of proteins and RNA, to the maximum in S phase, that have synthesis of DNA, of the cycle. / A ovinocultura atualmente ocupa um lugar de importância dentro dos criatórios de interesse zootécnico, sendo necessário o desenvolvimento de estudos na área de reprodução, relativos aos ovinos criados em clima tropical. Raças lanadas, em sua maioria possuem estacionalidade reprodutiva, ou seja, os ciclos são controlados pela diminuição da luminosidade da estação, já ovelhas mestiças geralmente possuem atividade reprodutiva geralmente observada durante todo o ano. A córtex ovariana possui os folículos e corpos lúteos em vários estádios de desenvolvimento, sustentada por tecido conjuntivo e fibras musculares lisas em meio a vasos e nervos. A medula ovariana é formada de tecido conjuntivo fibroelástico, nervos e artérias, compreendendo a parte mais interna do ovário. Em folículos antrais, o IGF estimula a proliferação, atividade e diferenciação das células da granulosa e a esteroidogênese. As regiões organizadoras de nucléolos (NORs) são alças de ácido desoxirribonucléico (DNA) responsáveis por sintetizar ácido ribonucléico ribossomal (RNAr) durante a intérfase, período de síntese protéica e de aumento de volume, tamanho e número de organelas da célula. É no nucléolo, localizado no núcleo celular, que há armazenamento e produção de RNAr, que ligados à várias proteínas irão formar os ribossomos. A técnica AgNOR avalia as NORs de cromossomos ativos durante o ciclo celular e o número ou área das NORs mostram essa atividade proliferativa, a partir da fase G1, de intensa produção de proteínas e RNA, até o máximo na fase S, de síntese do DNA, do ciclo.

Made available in DSpace on 2016-07-18T17:53:09Z (GMT). No. of bitstreams: 1 thais.pdf: 774762 bytes, checksum: aab7c8f9a2cbb52b94d59cd3cc962c07 (MD5) Previous issue date: 2011-10-24 / The sheep industry now occupies an important place of interest within the livestock farms, requiring the development of studies in the area related to sheep breeding on tropical climate. Most of the wool breed have reproductive seasonality, in other words, cycles are controlled by the fading light of the season, on the other hand crossbred sheep usually have already observed reproductive activity throughout the year. The ovarian cortex has follicles and corpus luteumin various stages of development, supported by connective tissue and smooth muscle fibers through the vessels and nerves. Te ovarian marrow is made up of fibroelastic tissue, nerves and arteries, being the innermost part of the ovary. In antral follicles, IGF stimulates the proliferation activity and differentiation of granulosa cells and steroidogenesis. The nucleolar organizer regions (NORs) are loops of deoxyribonucleic acid (DNA) responsible for synthesizing ribosomal ribonucleic acid (rRNA) during interphase, the period of protein synthesis and increase in volume, size and number of cell organelles. Its in the nucleolus, located in the cell nucleus, which has storage and production of rRNA, which will be linked to various proteins to form ribossomes. The AgNOR technique evaluates the NORs of active chromossomes during the cell cycle and the number or area of NORs show that proliferative activity, from the G1 phase of intense production of proteins and RNA, to the maximum in S phase, that have synthesis of DNA, of the cycle. / A ovinocultura atualmente ocupa um lugar de importância dentro dos criatórios de interesse zootécnico, sendo necessário o desenvolvimento de estudos na área de reprodução, relativos aos ovinos criados em clima tropical. Raças lanadas, em sua maioria possuem estacionalidade reprodutiva, ou seja, os ciclos são controlados pela diminuição da luminosidade da estação, já ovelhas mestiças geralmente possuem atividade reprodutiva geralmente observada durante todo o ano. A córtex ovariana possui os folículos e corpos lúteos em vários estádios de desenvolvimento, sustentada por tecido conjuntivo e fibras musculares lisas em meio a vasos e nervos. A medula ovariana é formada de tecido conjuntivo fibroelástico, nervos e artérias, compreendendo a parte mais interna do ovário. Em folículos antrais, o IGF estimula a proliferação, atividade e diferenciação das células da granulosa e a esteroidogênese. As regiões organizadoras de nucléolos (NORs) são alças de ácido desoxirribonucléico (DNA) responsáveis por sintetizar ácido ribonucléico ribossomal (RNAr) durante a intérfase, período de síntese protéica e de aumento de volume, tamanho e número de organelas da célula. É no nucléolo, localizado no núcleo celular, que há armazenamento e produção de RNAr, que ligados à várias proteínas irão formar os ribossomos. A técnica AgNOR avalia as NORs de cromossomos ativos durante o ciclo celular e o número ou área das NORs mostram essa atividade proliferativa, a partir da fase G1, de intensa produção de proteínas e RNA, até o máximo na fase S, de síntese do DNA, do ciclo.

Parapoxvirus ovis (PPVO) is known for its immunostimulatory capacities and has been successfully used to generate vector vaccines effective especially in non-permissive host species. Murine conventional and plasmacytoid dendritic cells (cDC and pDC) are able to recognize PPVO. The PPVO-sensing receptor on pDC is hitherto unknown. In this study we aimed to define the pattern recognition receptor responsible for the activation of murine pDC by inactivated and replication-competent PPVO. We show that PPVO-induced expression of type I and type III interferons, pro-inflammatory cytokines, and costimulatory CD86 by bone marrow-derived pDC but not cDC is blocked by chloroquine, an inhibitor of endosomal maturation. The activation of pDC is independent of viral replication and depends mainly on TLR9. Moreover, the use of phosphatidylinositol 3-kinase inhibitor wortmannin or C-Jun-N-terminal kinase inhibitor SP600125 results in significant reduction of PPVO-induced pDC activation. Taken together, our data identify endosomal TLR9 as PPVO-sensing receptor in pDC.

Thesis (MScAgric)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The application of assisted reproductive biotechnologies in sheep flocks is hampered by the susceptability of ovine sperm to cryodamage. There is still considerable scope in the improvement of cryopreservation protocols for ovine sperm to minimize the degree of damage to sperm during the cryopreservation process. Pre-cryopreservation processing has a definite effect on the survivability, motility, and fertilizing ability of sperm. Little information is however available on the potential contribution of the genetic make-up of rams, divergently selected for fecundity, on the ability of sperm to offer resistance to the damage caused by cooling, cryopreservation and thawing. The study aimed to investigate the influence of genetic selection for prolificacy (i.e. High Merino Line and Low Merino Line in terms of fecundity) on the ability of ovine sperm to offer resistance to cryodamage. The study investigated the effect of pre-cryopreservation processing by comparing motility and morphometry traits recorded for fresh- and post-thaw Merino ejaculated and epididymal sperm samples obtained form the High and Low lines, respectively. The effect of different sperm concentrations, equilibration periods and the addition or omission of seminal plasma from cryopreserved samples on the viability and morphometrical traits were also investigated. Ejaculate samples were collected by means of the artificial vagina (AV) method from 8 High Line rams and 7 Low Line rams. Epididymal samples were collected from 6 rams of each of the High and Low lines respectively, by recovering the epididymal sperm via aspiration from the cauda epididymides post mortem. Ejaculate samples were subjected to macroscopic and microscopic evaluation, and epididymal samples only to microscopic evaluation, for which the Sperm Class Analyzer® program was used for the evaluation of motility and morphometric measurements. Sperm motility recordings were captured at 100 frames per second. From findings of the study, it was concluded that genotype had no positive influence on the conception rate of the ewes mated to the High or Low Line rams, even though the rams from the two lines differed significantly in terms of their serving capacity. When sperm morphometry was evaluated for fresh ejaculate samples, the two lines differed significantly in terms of the morphometric traits elongation and ellipticity. Epididymal and ejaculated sperm obtained from Low Line rams had broader and rounder heads, compared to sperm obtained from High Line rams. When morphometry was assessed for sperm samples between the two methods of sperm recovery (collected with an AV or recovery via aspiration from the cauda epididymides of sacrificed rams), no morphometrical differences were observed. Significant differences were reported for the majority of the sperm motility traits (i.e. percentage motile, rapid-, medium-, slow swimming, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), and amplitude of the lateral head displacement (ALH)) recorded for ejaculated and epididymal sperm. The motility traits ALH and beat-cross frequency (BCF) analysed for epididymal sperm differed significantly between the two lines. When epididymal sperm were evaluated post-thaw, it became evident that the sperm obtained from the High Line rams had a larger acrosome surface cover when compared to that of the Low Line ram sperm. The addition of seminal plasma to epididymal samples did not result in an improvement of the preservation of sperm motility. It is known from the literature that cryopreservation causes a decrease in sperm head size. Head width was unaffected by cryopreservation with the addition of seminal plasma in this study, indicating a potential benefit with the use of seminal plasma in the cryopreservation protocol of epididymal ram sperm. The study compared two pre-processing techniques, i.e. the more time consuming swim-up technique (SUT) with a more time-efficient ‘flush technique’ (FT) to optimize the pre-processing protocol for motility assessment of sperm samples before cryopreservation of ram sperm. Comparison of the SUT and FT indicated that almost all of the motility parameters measured using the FT compared favourably with those obtained using the SUT. The results indicated that the FT can be used a more time-efficient technique to use for determining the motility of a sperm sample prior to cryopreservation. In conclusion, line differences associated with reproduction were observed in terms of the serving capacity of the rams, with selection for fecundity influencing the morphometric traits elongation and ellipticity for sperm obtained from the two lines. Future studies should be aimed at investigating morphometric traits of ovine sperm, to correlate it with fertilizing ability of sperm post-thaw and ensure optimal cryopreservation processing. / AFRIKAANSE OPSOMMING: Die toepassing van ondersteunende reproduksie tegnieke in skaaptroppe word bemoeilik deur die onvermoë van ram sperme om weerstand teen bevriesingskade te bied. Daar is nog baie ruimte vir die verbetering van die bevriesingsprotokolle vir skaap sperm om die omvang van bevriesingskade te verminder. Voor-bevriesing verwerking het dan 'n besliste uitwerking op die oorlewing, beweeglikheid en bevrugtingsvermoë, van skaap sperme. Min inligting is beskikbaar oor die potensiële bydrae van die genetiese samestelling van ramme wat uiteenlopend op grond van vrugbaarheid geselekteer is, op die vermoë van skaap sperme om weerstand te bied teen die skade wat deur verkoeling, diepbevriesing en ontdooiing, veroorsaak word. Die doelwit van die studie was om die invloed van genetiese seleksie vir fekunditeit (d.i. Hoë Merino Lyn en Lae Merino Lyn in terme van fekunditeit) op die vermoë van skaap sperme om weerstand teen bevriesingskade te bied, te ondersoek. Die studie het getoets wat die bevriesing proses se effek op epididimale sperme is, deur sperm motiliteit en -morfometrie te vergelyk tussen vars gekollekteerde sperme en sperm monsters na ontdooiing. Die effek van verskillende sperm konsentrasies, ekwilibrasie tydperke en die byvoeging of uitsluiting van seminale plasma op die lewensvatbaarheid en morfometriese eienskappe van Merino ramsperme is ondersoek in die studie. Geëjakuleerde monsters is versamel met behulp van 'n kunsmatige vagina (AV) van 8 Hoë Lyn en 7 Lae Lyn ramme. Epididimale monsters is verkry van 6 ramme van elk van die Hoë en Lae Lyne, deur middel van aspirasie van die sperme uit die cauda epididimii nadoods. Geëjakuleerde sperm monsters is met behulp van makroskopiese en mikroskopiese metodes geëvalueer, en epididimale sperm monsters slegs mikroskopies geëvalueer, met behulp van die Sperm Class Analyzer® program wat vir die evaluasie van beweeglikheid en morfometriese afmetings gebruik is. Sperm beweeglikheids opnames is opgeneem teen 100 raampies per sekonde. Die resultate van die studie het aangedui dat genotipe geen effek het op besetting van die ooie gepaar met die Hoë of Lae Lyn ramme gehad het nie, terwyl die dekvermoë aansienlik tussen ramme van die twee lyne verskil het. Wanneer die morfometriese eienskappe van vars geëjakuleerde sperme vergelyk was, het die lyne beduidend in terme van die morfometriese eienskappe van verlenging (elongation) en elliptisiteit verskil het. Die epididimale en geëjakuleerde sperme verkry vanaf die Lae Lyn ramme het ʼn breër en ronder kopvorm getoon as sperme wat verkry is van die Hoë Lyn ramme. Wanneer die morfometriese eienskappe van sperme versamel met die twee verskillende metodes (d.i. kunsmatige vagina of aspirasie vanuit die cauda epididimides) vergelyk was, is geen morfometriese verskille waargeneem nie. Die meeste sperm beweeglikheidseienskappe (d.i. persentasie beweeglike, vinnig-, medium- en stadig-swemmende sperme, VCL, VSL, VAP en ALH) van geëjakuleerde en epididimale sperme het verskil. Die beweeglikheidseienskappe amplitude van die laterale verplasing van die spermkop (ALH) en frekwensie waarmee sperm sy eie pad kruis (BCF), soos bepaal vir epididimale sperme, het beduidend tussen die twee lyne verskil. Met die evaluering van epididimale sperme na ontdooiing was dit duidelik dat sperme verkry van die Hoë Lyn ramme 'n groter mate van akrosoom-oppervlak gehad het, in vergelyking met sperme van die Lae Lyn ramme. Die byvoeging van seminale plasma by epididimale monsters het nie bygedra tot 'n verbetering van spermbeweeglikheid nie. Bestaande literatuur dui aan dat diepbevriesing 'n afname in die kopgrootte van sperme veroorsaak. In hierdie studie het die byvoeging van seminale plasma ʼn verandering in kopgrootte voorkom, wat dui op ʼn potensiële voordeel om seminale plasma in die bevriesingsprotokol van epididimale ramsperme in te sluit. Die studie het twee beweeglikheid bepalingstegnieke vergelyk om te bepaal of die tydrowende “opswem” tegniek (SUT) vervang kan word met 'n meer tyd-doeltreffende "spoel tegniek” (FT) in die voorbevriesing verwerking protokolle van ram sperme. Vergelyking van die twee tegnieke het aangedui dat die meeste van die kinematiese eienskappe van die FT gunstig met die waardes soos verkry met die SUT, vergelyk het. Resultate het getoon dat die FT parameters goed vergelyk met die beweeglikheid parameters van die SUT, dus kan dit aangeneem word dat die FT ʼn meer tyd-doeltreffende tegniek is wat vergelykbare sperm beweeglikheidsinligting oor skaap sperm monsters voor bevriesing sal verskaf. In samevatting is verskille in terme van die dekvermoë en op morfometriese vlak, meer spesifiek die eienskappe van verlenging (elongation) en elliptisiteit, tussen die twee lyne waargeneem. In toekomstige studies moet die morfometriese eienskappe van skaapsperme verder bestudeer word, asook die korrelasie daarvan met die bevrugtingsvermoë na ontdooiing bepaal om sodoende die diepbevriesing protokolle van skaapsperme te optimaliseer.

Memoria para optar al Título Profesional de Médico Veterinario / En el presente trabajo se desarrolló y validó un ensayo inmunoabsorbente ligado a un enzima (ELISA), usando antígeno lipopolisacárido rugoso (LPS-R) de la cepa RB51 de Brucella abortus para el diagnóstico de infección con B. ovis en ovinos. Se probaron 2 tipos de placas de poliestireno NUNC 69620 y Maxisorp, 2 tipos de tampones de distinto pH con 2 diferentes conjugados: policlonal anti IgG-ovina (Sigma A3415) y monoclonal anti IgG-caprino/ovino (Sigma A9452), las diluciones del antígeno y del conjugado del trabajo se obtuvieron mediante una titulación en tablero de ajedrez. El ensayo se validó utilizando la placa NUNC 69620 con una sensibilización del antígeno 1:50 en el tampón carbonato-bicarbonato y el uso del conjugado monoclonal, en 55 sueros de ovinos positivos a examen clínico y a la prueba de reacción en cadena de la polimerasa (PCR) y 338 sueros de ovinos provenientes de un rebaño libre de la enfermedad, confirmados por medio de fijación del complemento (FC). Para comparar las placas, las densidades ópticas (DO) fueron llevadas a porcentajes de positividad (PP), el punto de corte fue un 39,5% utilizando la metodología “Receiver-Operator Characteristic” (ROC), estimando la sensibilidad y la especificidad en un 92.7% y un 98,5% respectivamente. Estos resultados permiten establecer que el ELISA desarrollado sea una alternativa real, para el diagnóstico serólogico de B. ovis en el país

Comprendre de quelle façon les populations animales répondent aux conditions qui prévalent dans leur environnement revêt une grande importance. Les conditions climatiques et météorologiques sont une source importante de variabilité dans l'environnement et celles-ci ont des répercussions sur les espèces sauvages, en affectant leur physiologie, leur comportement et leurs ressources. Les ongulés alpins et nordiques font face à une succession annuelle de conditions environnementales favorables et défavorables, entraînant chez ceux-ci d'importants changements de masse saisonniers. Chez ces grands herbivores, la masse est importante puisqu'elle est positivement corrélée à la survie et à la reproduction. C'est pourquoi il est essentiel d'investiguer les paramètres affectant les changements de masse saisonniers. L'objectif principal de ma maîtrise était donc d'identifier et de mieux comprendre l'effet des conditions climatiques et météorologiques sur les changements de masse estivaux et hivernaux d'un ongulé alpin: le mouflon d'Amérique (Ovis canadensis). Pour atteindre cet objectif, j'ai utilisé les données du suivi à long terme de la population de mouflons de Ram Mountain, Alberta. Les mesures de masse répétées prises lors des captures permettent d'estimer la masse printanière et automnale des individus, ainsi que leur gain de masse estival et leur changement de masse hivernal. En affectant les coûts énergétiques de la thermorégulation et des déplacements et en influençant la végétation, les conditions climatiques et météorologiques peuvent avoir d'importantes conséquences sur les changements de masse des ongulés alpins. La température, les précipitations et un indice de climat global (le «Pacific Decadal Oscillation»; PDO) ont donc été utilisés afin de caractériser les conditions environnementales et d'investiguer les effets de ces variables sur les changements de masse saisonniers des individus de différentes classes d'âge et de sexe. Des températures froides au printemps ont engendré de plus grands gains de masse estivaux. Des printemps froids peuvent ralentir la maturation des plantes, engendrant une plus grande période où il est possible de s'alimenter de jeunes plantes nutritives, ce qui explique probablement cet effet positif des printemps froids. Cet effet doit toutefois être nuancé puisque les changements de masse hivernaux étaient également influencés par la température printanière, avec des printemps chauds menant à de plus faibles pertes de masse. Il semble que cet effet était dû à une apparition hâtive de la végétation, menant à une prise de masse des mouflons avant qu'ils ne soient capturés au printemps. Cela suggère qu'en affectant la disponibilité et la qualité de la végétation, les conditions printanières ont des répercussions à la fois sur le gain de masse estival, mais également sur les changements de masse hivernaux des mouflons. Le PDO au printemps a un effet positif important sur le gain de masse des adultes mâles lorsque la densité est faible. Des températures chaudes à l'automne engendrent de plus grands gains de masse pendant l'hiver chez les agneaux mâles (la plupart des agneaux gagnent de la masse l'hiver), potentiellement en augmentant la période possible de prise de masse pour ces jeunes individus. Les femelles de deux ans et les mâles adultes ont perdu plus de masse lors d'hivers avec beaucoup de précipitations et des températures froides, respectivement. Finalement, ce projet de recherche a permis d'identifier les variables climatiques et météorologiques clés affectant les changements de masse saisonniers d'un ongulé alpin. Cette étude a également mis en évidence des effets du PDO sur les changements de masse saisonniers, soulignant que de tels indices peuvent s'avérer utiles afin de les mettre en lien avec la variation phénotypique chez les espèces sauvages, et ce sans qu'il n'y ait nécessairement de fortes corrélations entre ces indices et les variables météorologiques locales.

<p><a>The hemotrophic mycoplasma<i>, Mycoplasma ovis</i>, is found in sheep and goats throughout the world. This pathogenic bacterium is capable of causing an acute, life-threatening infection as well as chronic or subclinical infections in these animals. The purposes of the present studies were to develop <i>M. ovis</i>-specific assays for detection of this hemoplasma, and to better understand infection dynamics within pregnant ewes and lambs. </a>The first study describes the development and validation of a SYBR^® Green quantitative PCR (qPCR) assay, which was subsequently used to determine the prevalence of <i>M. ovis</i> infection within a population of goats and to evaluate risk factors for infection. This highly sensitive and specific assay consistently detected as few as 10 copies of plasmid/reaction. Convenience-based sampling of 362 goats from 61 farms located in Indiana revealed a prevalence of infection of 18% (95% confidence interval (CI), 14% to 22%). Bacterial loads of <i>M. ovis</i> ranged from 1.05 x 10³ to 1.85 x 10⁵copies/mL of blood with a mean of 1.31 x 10⁴copies/mL of blood. The only risk factor associated with hemoplasma infection was the production use of the goat; dairy goats had a 3.3 fold increase compared with the prevalence in goats used for meat. This study not only demonstrates that <i>M. ovis</i> infection is common in goats in Indiana, but shows the variability of bacterial loads that can be found in chronically-infected animals. While sub-clinically infected goats may have a bacteremia, levels are characteristically less than 2.0 x 10⁵copies/mL.</p><p> The second project utilized a combination of cross-sectional and longitudinal studies to estimate the prevalence of <i>M. ovis</i> infection from a cohort of naturally-infected pregnant ewes, assess changes in their bacterial loads, and determine the incidence of <i>M. ovis</i> in lambs pre- and post-weaning. The prevalence of <i>M. ovis</i> infection in ewes was not found to be significantly different during pregnancy, and before and after weaning of the lambs, with prevalence estimates of 45% (95% CI, 23.1 – 68.5), 36% (95% CI, 17.9 – 57.4), and 44%, (95% CI, 24.4 – 65.1), respectively. Bacterial loads of the ewes from the cross-sectional study ranged from 10⁴to 10⁹copies/mL of blood, with the median bacterial load at 10⁵ copies/mL of blood. While higher bacterial loads are typical of an acute infection, none of the ewes in this study had overt clinical signs. The data suggest that <i>M. ovis</i> loads may be higher in pregnant sheep, particularly in ewes half-way through pregnancy. Most of the <i>M. ovis</i> infections in the study lambs were detected post-weaning which suggests that transplacental or transmammary infection of <i>M. ovis</i> are unlikely routes.</p><p> In the third study, a subset of <i>M. ovis</i> genes for use in a multi-locus sequence typing assay (MLST) were evaluated. Next-generation sequencing was performed to generate data from pooled DNA amplicons in order to identify single nucleotide polymorphisms (SNPs) of <i>M. ovis </i>from five genes. Evaluation of the quality and depths of coverage for the reads and SNPs indicated that the pooled DNA amplicons produced reads and SNPs having high quality and sufficient depth. This pooling technique is a cost-effective alternative to whole-genome sequencing. While the MLST has good discriminatory power and may be used to identify genetically distant and divergent clusters of <i>M. ovis</i> from different geographical origins, within a herd the discrimination power is low, which may hamper its usefulness in transmission studies. </p><p> The fourth and final study was the development of a loop-mediated isothermal amplification (LAMP) assay targeting the dnaK gene of <i>M. ovis</i>, with comparison of the assay to conventional PCR (cPCR). The metal ion indicator hydroxynaphthol blue (HNB) was added prior to the reaction, which allowed for visual detection of LAMP-positive samples as indicated by a color change from violet to sky blue. <i>Mycoplasma ovis</i> was consistently detected in 45 minutes with the LAMP assay at a reaction temperature of 64°C, with more infected sheep being detected than by cPCR. Therefore, the LAMP assay is fast and reliable in the detection of <i>M. ovis</i>. The developed LAMP assay may have applications in diagnostics, surveillance and disease management as well as prevalence studies. However, a more robust molecular technique is necessary for <i>M. ovis</i> isolate or stain discrimination to investigate transmission or disease spread in an outbreak.</p><p> </p><p> In conclusion, three new molecular tools for the detection of <i>M. ovis</i> in goats and sheep were developed as results of these studies. We have shown that the qPCR assay is an efficient tool for detection and quantification of <i>M. ovis</i> loads in blood from both of these species. On the other hand, the value of the LAMP assay is for reliable detection of infection (not quantification), especially in resource-limited situations. The five-locus MLST protocol developed herein, a typing assay based on the polymorphism of five gene sequences, is a laborious technique requiring DNA extraction, PCR amplification, purification and sequencing of target loci. The value of this technique is not as a routine diagnostic, but rather it may be used to better understand the genetic diversity of <i>M. ovis</i> and investigate strain variations. Most importantly, the scheme is sufficiently robust to allow direct genotyping of <i>M. ovis</i> in total blood DNA extracts without culture isolation. The MLST approach may prove useful as a tool for future investigations of transmission and disease spread. These studies have also expanded our understanding of the infection dynamics of <i>M. ovis</i> in pregnant sheep and lambs. It is shown herein that despite the high prevalence and sometimes high bacterial loads in pregnant ewes, <i>M. ovis</i> does not appear to be transmitted to the lambs in utero or during the perinatal period. The lambs become infected mostly after weaning; this may suggest a protective effect during the pre-weaning period and/or subsequent exposure/infection from their environment. </p><br>

Psoroptic mange, caused by the Astigmatid mite Psoroptes ovis, is an ectoparasitic disease of significant economic importance to agriculture on a global scale and poses a serious welfare concern. With the current chemotherapeutic controls considered unsustainable, there is pressing need for novel control strategies. RNA interference has been proposed as a potential high throughput approach for the identification of novel therapeutic targets with high specificity, speed and at a relatively low cost compared to the existing methods. The presence of the components of the RNA interference (RNAi) pathway in P. ovis was first confirmed through in silico analyses of the P. ovis transcriptome and, following development of a non-invasive immersion method of double stranded RNA (dsRNA) delivery, gene silencing by RNAi was demonstrated in P. ovis. Statistically-significant reduction of transcript level was measured for the three genes targeted: P. ovis mite group 2 allergen (Pso o 2), P. ovis mu class glutathione S-transferase (PoGST-mu1) and P. ovis beta tubulin (Poβtub). This is the first demonstration of gene silencing by RNAi in P. ovis and provides a key mechanism for mining transcriptomic and genomic datasets in the future for novel targets of intervention against P. ovis. The first assessment of gene silencing was also performed in two related Astigmatid mites of high medical importance; the European house dust mite Dermatophagoides pteronyssinus and the scabies mite Sarcoptes scabiei. A statistically-significant reduction in expression of a D. pteronyssinus mu class glutathione S-transferase (DpGST-mu1) transcript was observed. No significant reduction in expression of a S. scabiei mu class glutathione S-transferase (SsGST-mu1) transcript was observed. Additionally, microRNAs (miRNAs) from the related miRNA pathway were identified in a P. ovis small RNA sample and were sequenced and annotated.

This investigation explored the utility of mean osteon area, perimeter, and circularity as parameters for distinguishing between human and non-human bone. Although species of origin can be readily ascribed to undamaged remains, this evaluation can become difficult, if not impossible, when bone macrostructure has been obscured through taphonomic processes such as thermal alteration or extreme fragmentation. If mature Ovis aries, domestic sheep, and human osteon metrics area are significantly different, then osteon metrics can be useful parameters for distinguishing between these tissues and, thus, determining the species of origin for a bone sample of unknown provenience. This investigation consisted of histological analysis of cortical bone from 35 O. aries rib samples. O. aries samples were acquired from Riverslea Farm in Epping, New Hampshire (n = 30) and Cedar Ledge Farm in Somers, Connecticut (n = 5). Mean osteon area, perimeter, and circularity were assessed by manually tracing calibrated digital images of rib cross-sections using the ImageJ software from the National Institute of Health (NIH, Bethesda, MD). The results presented here indicate (1) that there are statistically significant histomorphometric differences between species, specifically goat and sheep, (2) that there are significant histomorphometric differences based on the anatomical, intra-rib sampling location, and (3) that the osteon metrics discussed here are may be poor parameters by which to ascribe the species of origin for remains with no known provenience, based on comparisons with findings from other publications.

Herbivory is one major force accelerating aspen decline in North America, but it is unclear why herbivores prefer certain aspen stands over others, or over other plant species in the understory. In this dissertation, I determined the influence of nutrients and plant secondary compounds (PSC), physiological state, chemical composition, and prior experience on aspen preference by sheep in controlled pen experiments. In addition, I explored the relationship between herbivory, regeneration, recruitment, and other landscape elements for specific aspen stands within Wolf Creek Ranch in northern Utah using biomass and chemical composition of the understory and chemical defenses of juvenile aspen trees (i.e., the foodscape). Aspen intake was enhanced when lamb diets contained a high crude protein to energy ratio or when the basal diet contained a low density of energy. Intake was depressed as concentrations of PG (phenolic glycosides) increased in aspen leaves or when lambs were fed a high energy to protein ratio. The effects of nutrients on aspen intake were greater when phenolic glycosides in aspen were present at low concentrations. However, when given a choice between aspen leaves of high or low PG content, lamb preference depended more on aspen nutrient and mineral availability, or on prior diet, than on defense chemistry. On the landscape, I found that stands at low elevations with low abundance of nutrients in the understory are more likely to experience less regeneration and recruitment than those growing within nutrient-rich sites. Aspen browsing was negatively correlated with PG content in aspen stands, and elk presence (measured via fecal pellets) was negatively correlated with abundance of understory protein. In conclusion, aspen herbivory appears to be controlled by the interplay between types and amounts of nutrients offered by the landscape and the chemical composition of aspen stands. A clear assessment of these variables on the landscape, i.e., the foodscape, will aid in the development of novel management strategies aimed at providing nutrients (e.g., through supplements, introduced forages) at strategic locations in order to reduce aspen herbivory within at-risk aspen stands.