Recently, examination of deoxyribonucleic acids in water samples (environmental DNA or eDNA) has shown promise for identifying fish species present in water bodies. In water, eDNA arises from bodily secretions such as mucus, gametes, and feces. I investigated whether eDNA can be effective for characterizing fish presence, relative abundance, biomass, and species composition in a large Arizona reservoir (Theodore Roosevelt Lake) and 12 small Arizona (<24 ha) waterbodies. Specifically, I compared fish presence, relative abundance (catch per unit effort [CPUE]), biomass (biomass per unit effort [BPUE]), and species composition measured through eDNA methods and established American Fisheries Society (AFS) standard sampling methods in Theodore Roosevelt Lake and 12 small waterbodies. Environmental DNA sampling resulted in detection of Gizzard Shad Dorosoma cepedianum at a higher percentage of sites than boat electrofishing, both in spring and fall. Contrarily, gill nets detected Gizzard Shad at more sites than eDNA for both spring and fall sampling in Lake Roosevelt. Boat electrofishing and gill netting detected Largemouth Bass Micropterus salmoides at more sites than eDNA, with the exception of fall gill net sites which equally detected Largemouth Bass at sites within Lake Roosevelt. Environmental DNA detected Largemouth Bass and Bluegill Lepomis macrochirus at more Arizona small lakes than detection with established gear methods. I observed no relationship between relative abundance and biomass of Largemouth Bass and Gizzard Shad measured by established methods and their DNA copies at individual sites or by lake section in Lake Roosevelt. Likewise, I found no relationship between relative abundance and biomass of Largemouth Bass and Bluegill measured by established methods and their DNA copies across 12 small waterbodies. Plot analysis conceivably illustrated that reservoir-wide catch composition (numbers and total weight of fish [g]) achieved through a combination of gear types (boat electrofishing + gill netting) for Largemouth Bass and Gizzard Shad was slightly similar to the proportion of total eDNA copies of each species for both spring and fall field sampling. Likewise, spring and fall gill net surveys somewhat portrayed total catch composition (numbers and total weight of fish [g]) of Largemouth Bass and Gizzard Shad similar to the proportion of total eDNA copies of each species. The exception was the total lack of similarity illustrated between proportions of fish caught in spring and fall boat electrofishing and total eDNA copies of each species in Lake Roosevelt. However, the deceptive similarity of all the plots were not present in the chi-square analysis with the exception of fall gill net surveys in Lake Roosevelt. In addition, eDNA did reflect the relative proportions of Largemouth Bass and Bluegill in total catch composition in some, but not all of 12 small Arizona waterbodies. The ease of eDNA sampling over established fish sampling makes it appealing to natural resource managers. Compared to current established fish sampling methods, eDNA sampling can be less laborious, less time consuming, and more cost effective. Environmental DNA sampling may be useful in sites that have difficult access such as remote sites. However, evaluation of eDNA is necessary to identify limitations and benefits in fish monitoring programs. Furthermore, field sampling protocols, filtration, DNA extraction, primer design, and DNA sequencing methods need further refinement and testing before incorporation into standard fish sampling surveys.

Metropolitan mosquitoes: Understanding urban livestock keeping and vector-borne disease in growing tropical sites – The potential of sustainable control methods and the risks for emergence to Sweden.

<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>

Abstract Introduction This study evaluated the effect of the polymer polyvinylalcohol-carbazate (PVAC) on the bacteria Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa. PVAC is a polymer with a carbazate moiety that neutralizes free aldehydes and has shown great promise in stabilizing erythrocytes during long term storage. It has also been shown to reduce intraperitoneal adhesions after trauma. For this study, two Gram positive and two Gram negative bacteria strains were used with PVAC to evaluate its effect. Materials and methods PVAC was obtained from the research team at Ångström Laboratory, Uppsala. The bacteria were obtained from Clinical Microbiology and Hospital Hygiene, Academic Hospital, Uppsala. The methods used were spectrophotometric assessment of bacteria growth, use of FITC-conjugated PVAC to study adherence to bacteria, use of FITC-antibodies to study PVAC’s effect on bacterial adherence to erythrocytes and a qPCR for quantification of E. coli. Results and discussion PVAC displayed a clear effect of inhibition of bacteria growth in the study as shown by use of spectrophotometric assessment. Trials with FITC-PVAC showed that the polymer adheres directly to the bacteria, displaying a possible function of its inhibitory properties. The qPCR assay was able to detect the bacteria in all the dilutions used. Introduction This study evaluated the effect of the polymer polyvinylalcohol-carbazate (PVAC) on the bacteria Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa. PVAC is a polymer with a carbazate moiety that neutralizes free aldehydes and has shown great promise in stabilizing erythrocytes during long term storage. It has also been shown to reduce intraperitoneal adhesions after trauma. For this study, two Gram positive and two Gram negative bacteria strains were used with PVAC to evaluate its effect. Materials and methods PVAC was obtained from the research team at Ångström Laboratory, Uppsala. The bacteria were obtained from Clinical Microbiology and Hospital Hygiene, Academic Hospital, Uppsala. The methods used were spectrophotometric assessment of bacteria growth, use of FITC-conjugated PVAC to study adherence to bacteria, use of FITC-antibodies to study PVAC’s effect on bacterial adherence to erythrocytes and a qPCR for quantification of E. coli. Results and discussion PVAC displayed a clear effect of inhibition of bacteria growth in the study as shown by use of spectrophotometric assessment. Trials with FITC-PVAC showed that the polymer adheres directly to the bacteria, displaying a possible function of its inhibitory properties. The qPCR assay was able to detect the bacteria in all the dilutions used.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-10-14T19:55:31Z No. of bitstreams: 2 Tese - Hugo Delleon da Silva - 2013.pdf: 14374996 bytes, checksum: d653dbc3306ce002b867a103b5a20bb4 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-10-16T18:33:50Z (GMT) No. of bitstreams: 2 Tese - Hugo Delleon da Silva - 2013.pdf: 14374996 bytes, checksum: d653dbc3306ce002b867a103b5a20bb4 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-16T18:33:50Z (GMT). No. of bitstreams: 2 Tese - Hugo Delleon da Silva - 2013.pdf: 14374996 bytes, checksum: d653dbc3306ce002b867a103b5a20bb4 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-10-10 / Human adenovirus (HAdV) is indicated as a viral biomarker of water quality. Thus, studies that show or even the occurrence of these interactions in water are of great importance, since these studies are still scarce. The aims of these studies were: (i) to detect, quantify and molecularly characterize the HAdV serotypes that can to circulate in the water supply treatment in the city of Goiania; (ii) evaluate the infectivity and recovery of genomic copies (GC) of HAdV-5 in simulated condition with solids derived from tropical soils and under controlled conditions of the pH and the presence of organic matter (OM); (iii) establish a mathematical equation to evaluate the recovery rate of virus genome copies in simulated conditions with clay soils. Thus, in the second half of 2012, we collected sample water in a volume of 5 L of 4 treated water reservoir and their respective points in the distribution network in the city of Goiania, totaling 80 samples. The samples were concentrated, quantified by qPCR and sequenced. Altogether 76.6% (100 - 109 CG/mL) and 37.5% (101 - 108 CG/mL) of the samples were positive for the reservoir and their respective points in the distribution network respectively. Therefore, Goiânia’s treated water is contaminated with a high number of HAdV type C. In the study of the interaction of HAdV-5 with the solid (sediments), a hydromorphic soil sample was divided into two parts: soil organic matter (WOM) and soil less organic matter (LOM). Then, it was added separately 5, 25 and 50 mg of soil WOM and LOM in sterile polypropylene tubes of 50 mL, added ultrapure water and adjusted the pH to 6.0 and 8.0. Then was added 1 mL of viral aliquot and the volume made up to 50 mL. The tubes in replica were shaken at 150 rpm for 1h at 24 °C followed by decantation. The viral genome was quantified (GC/mL) by Real-Time PCR and the Infectivity by Assay Plate Lysis. Water with solids promoted a reduction in the number of GC/mL and viral infectivity. The OM did not affect the recovery of GC/mL (p> 0.05). However, the OM was harmfulto to the infectivity of the virus, with a reduction of 2 log10 of Plaque Forming Units per milliliter (PFU/mL), when compared with treatments LMO. The acidic pH is unfavorable to virus inactivation, and the clay is the main element responsible for the interaction of HAdV-5. The mathematical equation is useful in assessing the recovery of viral genomic copies in clay solutions. These data can offer support in ecoepidemiological studies of viral inactivation or water treatment. / O adenovírus humano (HAdV) é indicado como um biomarcador viral para a análise de qualidade da água. Assim, estudos que evidenciam a ocorrência ou mesmo as interações destes em água são de suma importância, já que as pesquisas na área são inexpressivas. Neste contexto, foram objetivos do estudo: (i) detectar, quantificar e caracterizar molecularmente os HAdVs circulantes no sistema de abastecimento de água tratada da cidade Goiânia; (ii) avaliar a infecciosidade e a recuperação de cópias genômicas (CG) de HAdV-5 em soluções simuladas com sólidos tropicais e em condições controladas de pH e presença de matéria orgânica (MO) e (iii) estabelecer uma equação matemática para avaliar a taxa de recuperação de cópias genômicas virais em condições simuladas com solos argilosos. Assim, no segundo semestre de 2012, foram coletadas amostras de água tratada (5 L) em 4 reservatórios e em seus respectivos pontos na rede de distribuição de Goiânia, totalizando 80 amostras. As amostras foram concentradas, quantificadas por PCR em Tempo Real Quantitativa (qPCR) e parte destas sequenciadas. Ao todo, 76,6% (100 - 109 CG/mL) e 37,5% (101 - 108 CG/mL) das amostras foram positivas para os reservatórios e seus pontos na rede de distribuição respectivamente. Portanto, a água tratada de Goiânia está contaminada por um elevado número de HAdV tipo C. Quanto ao estudo com os sólidos, amostra de um solo hidromórfico foi subdividida em duas partes: solo com matéria orgânica (CMO) e solo sem matéria orgânica (SMO). Foi adicionado separadamente 5, 25 e 50 mg de solo CMO e SMO em tubos de polipropileno estéreis de 50 mL, adicionado água ultrapura e o pH das soluções ajustado para 6,0 e 8,0. Em seguida, foi adicionado 1 mL de alíquota viral e o volume foi completado para 50 mL. Os tubos em réplica foram agitados a 150 rpm por 1h a 24 ºC e decantados por 1h. O genoma viral foi quantificado (CG/mL) por qPCR e a Infecciosidade por Ensaio em Placa de Lise. A água com sólidos promoveu uma redução na recuperação das CG/mL e na infecciosidade viral. A MO não influenciou na recuperação das CG/mL (p>0,05), todavia, foi predudicial à infecciosidade viral, com diminuição de 2 log10 de Unidades Formadoras de Placa por Mililitro (PFU/mL), quando comparado com os tratamentos SMO. O pH 6,0 desfavorece a inativação e a argila é o principal elemento responsável pela interação do HAdV-5. A equação matemática é útil na avaliação da recuperação das cópias genômicas virais em soluções argilosas. Estes dados podem auxiliar em estudos eco-epidemilógicos, de inativação viral ou tratamento da água.

A cultura da cana-de-açúcar é a segunda maior cultura em valor de produção agrícola do país. Nos últimos anos ocorreu uma alteração no cenário canavieiro passando de colheita manual de cana queimada para mecanizada de cana crua. A colheita mecanizada da cana-de-açúcar acarreta muitos benefícios ambientais, porém o tráfego intenso de maquinários resulta na compactação do solo. Desse modo, buscamos avaliar como o efeito das alteração física do solo causado pelo tráfego de máquinas durante a colheita da cana interfere na composição das comunidades microbianas, especialmente as que participam das transformações do nitrogênio. As parcelas experimentais foram divididas em colheita manual (PDTR) e colheita mecanizada (PD). Todos os demais manejos e tratos culturais foram iguais, isolando a influência do impacto do maquinário durante a colheita. Para a avaliação da microbiota realizamos análise da estrutura da comunidade (DGGE ou T-RFLP) e análise de abundância (qPCR) de bactérias, fungos, arquéias, e do ciclo do nitrogênio envolvendo os genes marcadores para a nitrificação (amoA - AOA e AOB), fixação de nitrogênio (nifH) e desnitrificação (nirK, nirS, nosZ clado I e II). Observamos apenas a diferença dos parâmetros físicos na camada superficial por meio da resistência a penetração. A alteração no perfil da comunidade de bactérias e arquéias mostra que ambas são responsivas ao tratamento com tráfego do maquinário. Em relação aos microrganismos envolvidos nas transformações de nitrogênio, AOA foi altamente responsiva ao impacto do tráfego agrícola em ambos os tipos de textura, mas teve diferença na abundância apenas no solo arenoso. O gene nifH apresentou diferença na diversidade em solo argiloso e diferença de abundância em solo arenoso. E por fim, sugerimos que o gene nosZ em subsuperfície pode indicar uma possível campactação antes dos parâmetros físicos do solo. / In Brazil, sugarcane is the second most value of agricultural production. Lately, the harvest management in sugarcane fields has changed from manual harvesting methods with pre-harvest burns to mechanical harvests with green cane (unburnt harvest). The mechanized harvest brings many ambiental benefits. However, the intense traffic due to agricultural machines over the years is resulting in soil compaction. The purpose of this work was to evaluate how the physical effect of mechanized harvesting can interfere with the composition of soil microbial community, specially the microbial involved in the nitrogen cycle. Experimental plots were divided into manual harvest (PDTR) and mechanized harvest (PD). All aspects of crop management are the same, so we were able to study the impact of mechanical harvester traffic during the harvest in isolation. To evaluation of the microbiome we analyzed community structure (DGGE ou T-RFLP) along with their abundance (qPCR) for soil bacterial, fungal and archaeal, and microorganisms involved in the transformation of nitrogen that we used gene markers for nitrification (amoA - AOA and AOB), nitrogen fixation (nifH) and denitrification (nirK, nirS, nosZ clade I and II). We observed the difference between physical parameters only in topsoil by soil penetration resistance. Our results indicated structured community changes of bacterial and archaeal showed us that both are responsive to treatment of machinery traffic. Microorganisms involved in the nitrogen cycle, our results presented that AOA is highly responsive to the impact of agricultural traffic on both soil texture, but we observed the difference in abundance only in sandy soil. The nifH gene was different on community structure in clay soil and on abundance in sandy soil. Moreover, we suggest that nosZ gene could indicate a possible soil compaction before the physical parameters in a layer between 20 and 40 cm.

A cultura da cana-de-açúcar é a segunda maior cultura em valor de produção agrícola do país. Nos últimos anos ocorreu uma alteração no cenário canavieiro passando de colheita manual de cana queimada para mecanizada de cana crua. A colheita mecanizada da cana-de-açúcar acarreta muitos benefícios ambientais, porém o tráfego intenso de maquinários resulta na compactação do solo. Desse modo, buscamos avaliar como o efeito das alteração física do solo causado pelo tráfego de máquinas durante a colheita da cana interfere na composição das comunidades microbianas, especialmente as que participam das transformações do nitrogênio. As parcelas experimentais foram divididas em colheita manual (PDTR) e colheita mecanizada (PD). Todos os demais manejos e tratos culturais foram iguais, isolando a influência do impacto do maquinário durante a colheita. Para a avaliação da microbiota realizamos análise da estrutura da comunidade (DGGE ou T-RFLP) e análise de abundância (qPCR) de bactérias, fungos, arquéias, e do ciclo do nitrogênio envolvendo os genes marcadores para a nitrificação (amoA - AOA e AOB), fixação de nitrogênio (nifH) e desnitrificação (nirK, nirS, nosZ clado I e II). Observamos apenas a diferença dos parâmetros físicos na camada superficial por meio da resistência a penetração. A alteração no perfil da comunidade de bactérias e arquéias mostra que ambas são responsivas ao tratamento com tráfego do maquinário. Em relação aos microrganismos envolvidos nas transformações de nitrogênio, AOA foi altamente responsiva ao impacto do tráfego agrícola em ambos os tipos de textura, mas teve diferença na abundância apenas no solo arenoso. O gene nifH apresentou diferença na diversidade em solo argiloso e diferença de abundância em solo arenoso. E por fim, sugerimos que o gene nosZ em subsuperfície pode indicar uma possível campactação antes dos parâmetros físicos do solo. / In Brazil, sugarcane is the second most value of agricultural production. Lately, the harvest management in sugarcane fields has changed from manual harvesting methods with pre-harvest burns to mechanical harvests with green cane (unburnt harvest). The mechanized harvest brings many ambiental benefits. However, the intense traffic due to agricultural machines over the years is resulting in soil compaction. The purpose of this work was to evaluate how the physical effect of mechanized harvesting can interfere with the composition of soil microbial community, specially the microbial involved in the nitrogen cycle. Experimental plots were divided into manual harvest (PDTR) and mechanized harvest (PD). All aspects of crop management are the same, so we were able to study the impact of mechanical harvester traffic during the harvest in isolation. To evaluation of the microbiome we analyzed community structure (DGGE ou T-RFLP) along with their abundance (qPCR) for soil bacterial, fungal and archaeal, and microorganisms involved in the transformation of nitrogen that we used gene markers for nitrification (amoA - AOA and AOB), nitrogen fixation (nifH) and denitrification (nirK, nirS, nosZ clade I and II). We observed the difference between physical parameters only in topsoil by soil penetration resistance. Our results indicated structured community changes of bacterial and archaeal showed us that both are responsive to treatment of machinery traffic. Microorganisms involved in the nitrogen cycle, our results presented that AOA is highly responsive to the impact of agricultural traffic on both soil texture, but we observed the difference in abundance only in sandy soil. The nifH gene was different on community structure in clay soil and on abundance in sandy soil. Moreover, we suggest that nosZ gene could indicate a possible soil compaction before the physical parameters in a layer between 20 and 40 cm.

La paratuberculose est une maladie enzootique contagieuse des ruminants due à Mycobacterium avium ssp. paratuberculosis (Map). La longue période d'incubation et les faibles performances diagnostiques des tests limitent l'efficacité des plans de maîtrise. Les grands effectifs des troupeaux ovins limitent l'approche par dépistage individuel exhaustif, et les plans de maîtrise s'orientent vers la vaccination, dont les effets n'ont pas été évalués dans les troupeaux français. Á partir d'échantillons de sérum et de fèces de près de 4000 brebis issues de 30 élevages du Lot, les performances diagnostiques de deux trousses sérologiques ELISA et d'une qPCR sur fèces ont été estimées par des modèles à classes latentes bayésiens et fréquentistes. Nos résultats confirment la faible sensibilité et le défaut de spécificité des sérologies ELISA pour la détection des ovins infectés ; la qPCR présentant de meilleures performances diagnostiques. Par ailleurs nous avons évalué les performances diagnostiques relatives des ELISA et de la qPCR appliquées à des mélanges d'échantillons. Dans les deux cas les animaux forts répondeurs étaient détectés de façon systématique, les animaux faiblement positifs étant détectés de façon moins constante. Sur la base de simulations, nous avons évalué les performances des stratégies de dépistage et de suivi basées sur les analyses de mélanges d'échantillons à l'échelle des troupeaux. Alors que la sérologie ELISA est apparue insuffisamment spécifique, l'analyse de mélange de fèces par qPCR semble être une approche prometteuse, permettant de détecter des faibles prévalences d'infection. Enfin les travaux menés dans les troupeaux vaccinés ont précisé dans quelles mesures leur situation épidémiologique pouvait être approchée par l'emploi d'analyses en mélanges. / Paratuberculosis is a contagious enzootic disease in ruminants caused byMycobacteriumavium ssp. paratuberculosis (Map). The long incubation period and the low informative values of antemortem diagnostic tests limit the effectiveness of control plans. In large sheep flocks, exhaustive individual testing is unfeasible and control plansmainly focus on vaccination, the effects of which have not yet been evaluated in French flocks. Using blood and feces samples from nearly 4000 ewes in 30 sheep flocks from the French department of Lot, the diagnostic performances of two serum ELISA and one fecal qPCR kits were estimated using bayesian and frequentist latent class modeling. Our results confirm the low sensitivity and non-perfect specificity of serum ELISA for the detection of subclinically infected animals, while the diagnostic performances of fecal qPCR were better. We also evaluated the relative diagnostic performances of pooled-sample analysis for both tests. Highly qPCR/ELISA positive samples were invariously detected while low positive ones were associated with lower detection rates. The flock-level epidemiological performances of screening strategies based on pooled-sample analysis were evaluated by simulation studies. Pooled serum ELISA appeared lowly specific. Conversely, pooled fecal qPCR appeared promising, allowing the detection of low infection prevalence. Finally, the work carried out in the vaccinated flocks made it possible to better know their epidemiological status and to specify to what extent it could be approached using pooled-sample analysis.

Nonnative species that cause damage to ecosystems to which they are introduced are considered in-vasive. Restoration of the original ecosystem after an invasive population has established is expensive and difficult but more likely to succeed when invasions are discovered early. Containment efforts to prevent the spread of known invasions also benefit from earlier knowledge of invaded sites. Environ-mental DNA (eDNA) techniques are emerging as a tool that can identify invasive species at a distinctly earlier time point than traditional methods of detection. I collected water samples from eight sites not known to be invaded by the freshwater New Zealand mud snail (NZMS). After filtering these samples to collect eDNA, I used a species-specific probe with qPCR to identify NZMS eDNA. I found evidence for NZMS invasion at five of the eight sites, with later physical confirmation of mud snails at one of these sites. This study is the first example of successful application of eDNA to detect new invasions of the freshwater New Zealand mud snail, setting the stage for further monitoring of at-risk sites to de-tect and control new invasions of this destructive snail.

Unregulated use of growth promoting antibiotics like Tetracyclines in agricultural feeds is becoming an increasing problem in antibiotic resistance. Undigested antibiotics leads to significant concentrations in livestock waste. These concentrations provide continuous selection pressure for the development of antibiotic resistance genes in the environment. Antibiotic resistance related deaths are projected to surpass cancer related deaths by 2050 making antibiotic resistance a pressing public health issue. The purpose of this study is to determine the abundance and persistence of tetracycline (tet) resistance genes in swine waste over a period of 100 days in an anaerobic digester system. Tet(A), tet(B), tet(G), tet(M), tet(O), tet(Q), and tet(W) were quantified by quantitative polymerase chain reaction after DNA extraction. Primers that target ribosomal protection proteins and efflux proteins were used. Antibiotic resistance genes decreased from day one but were found to be present throughout the study.