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Managing turf for high aesthetic value is costly. Such management usually involves mowing, disease prevention, insect control, and weed control. Mowing is the most expensive practice on golf courses and annual bluegrass (Poa annua L) is the most challenging weed problem in professional turf. The plant growth regulators trinexapac-ethyl and paclobutrazol are commonly used in VA for these two costly and challenging jobs. Prohexadione calcium (PC) is an experimental chemical that inhibits the same enzyme (3ß-hydroxyalase) as trinexapac-ethyl and may selectively suppress annual bluegrass. Experiments were conducted at the Virginia Tech Turfgrass Research Center and Glade Road Research Facility to determine the PC rate required to reduce clipping biomass of four turfgrass species as effectively as trinexapac-ethyl. Prohexadione calcium reduced clipping biomass of bermudagrass (Cynodon dactylon (L.) Pers.), Kentucky bluegrass (Poa pratenis L.), perennial ryegrass (Lolium perenne L.), and zoysiagrass (Zoysia japonica Steud.) equivalent to trinexapac-ethyl at 0.70, 0.22, 0.60, and 0.27 kg a.i./ha -1, respectively. Further experiments conducted at three locations across Virginia determined that PC was comparable to paclobutrazol for annual bluegrass suppression. Since turfgrass response to PC was different between annual bluegrass, Kentucky bluegrass, and perennial ryegrass, 14C labeled PC was used to assess absorption, translocation, and metabolism of PC between annual and Kentucky bluegrass, creeping bentgrass (Agrostis stolonifera L.), and perennial ryegrass. Annual and Kentucky bluegrass absorbed more PC than creeping bentgrass or perennial ryegrass and partially explained the selectivity between these species. Translocation and metabolism of PC did not differ between species. Our final objective launched experiments characterizing possible resistance to acetolactate synthase (ALS) inhibiting herbicides in annual bluegrass. Several selective herbicides for annual bluegrass control inhibit ALS. Since many weeds have developed resistance to ALS-inhibiting herbicides, the ALS gene in annual bluegrass was sequenced and derived amino acid sequences were at least 87% similar to other previously sequenced grass species. This sequencing data will be used in future experiments to predict the likelihood of ALS resistance in annual bluegrass. / Ph. D.

O parvovírus humano B19 é um vírus não envelopado, que contém uma fita simples de DNA. Seu genoma é altamente conservado, com 98 a 99% de similaridade entre os isolados. Durante a última década, variantes do parvovírus B19, gênero Erythrovirus, foram descritas apresentando variabilidade genética de 11 a 14. Uma nova classificação foi proposta, dividindo o gênero em três diferentes genótipos (1, 2, 3a e 3b). Para avaliar a diversidade genética e o papel epidemiológico dos eritrovírus, 892 amostras brasileiras e chilenas, foram investigadas para a presença de DNA viral. As amostras positivas foram seqüênciadas e genotipadas através da analise da região VP1/VP2. Os resultados mostraram predominância do genótipo 1 (89 amostras), seguido do genótipo 3 (1 amostra), nas amostras brasileiras, enquanto que nas amostras chilenas, apenas o genótipo 1 foi observado (24 amostras). A única variante detectada, a amostra BR543, apresentou variabilidade de aproximadamente 13%, quando comparada ao B19, sendo classificada como genótipo 3, subtipo 3b. / Human Parvovirus B19 is a non-enveloped, ssDNA virus. The genome is highly conserved, showing 98 to 99% of nucleotide similarity between the isolates. During the last decade, parvovirus B19 variants, genus Erythrovirus, were described showing 11 to 14 %. A new classification was suggested, dividing the genus in three different genotypes (1, 2, 3a and 3b). To evaluate the epidemiology and the erythrovirus genetic diversity, 892 brazilians and chileans samples, were investigated for the virus DNA presence. The positive samples were sequenced and genotyped (VP1/VP2). The results showed the prevalence of genotype 1 (69 samples), followed by genotype 3 (1 sample), in the brazilians samples, and in the Chilean samples only genotype 1 was observed (24 samples). The variant detected, BR543, showed 13% of divergence when compared to B19, classified as genotype 3, subtype 3b.

Pienarūgščių bakterijų paieška ir jų identifikavimas migruojančių didžiųjų ančių (Anas platyrhynchos) žarnyne naudojant dalinių 16S rRNR geno sekų analizę ir kultivavimu paremtus metodus Klaidas VARNA Vilniaus Universiteto Ekologijos Institutas, Hidrobiontų Ekologijos ir Fiziologijos Laboratorija bei Populiacinės Genetikos Laboratorija, Akademijos-2, Vilnius-21, 08412, Lietuva. Šiame tyrime pavasarinių ir rudeninių didžiųjų ančių (Anas platyrhynchos) migrantų iš Nemuno deltos virškinamojo trakto pieno rūgšties bakterijų įvairovė buvo ištirta naudojant molekulinius metodus (polimerazės grandininės reakcijos amplifikacija ir dalinių 16S rRNR geno sekų sekvenavimas) ir kultivavimu paremtus metodus. Migruojančių didžiųjų ančių (Anas platyrhynchos) pieno rūgšties bakterijų paieška buvo atlikta pirmą kartą. Rudeniniai didžiųjų ančių migrantai plonojo žarnyno sienelėse (1.2×107 iki 2.1×107 k.f.v./g) ir jų turinyje (nuo 3.4×107 iki 1.1×108 k.f.v./g) turi didesnį pieno rūgšties bakterijų skaičių nei pavasariniai migrantai (atitinkamai nuo 3.2×106 iki 4.8×106 k.f.v./g ir nuo 1.0×107 iki 2.2×107 k.f.v./g). Tiek rudeninių tiek ir pavasarinių didžiųjų ančių migrantų plonojo žarnyno sienelėse ir jų turinyje dominavo kokinės pieno rūgšties bakterijų formos (atitinkamai 65% ir 83.5% bei 81.4% ir 91.6%), o lazdelių buvo mažiau (atitinkamai 35% ir 16.5% bei 18.6% ir 8.4%). Manoma, kad minėtus skirtumus įtakoja keli veiksniai: ilgai trunkanti migracija, perėjimo periodas, skirtingas maistas ir... [toliau žr. visą tekstą] / Identification of lactic acid bacteria in the migrant mallard ducks Anas platyrhynchos intestinal tract by partial 16S rRNA gene sequence analysis and using culture-based techniques Klaidas VARNA Institute of Ecology of Vilnius University, Laboratory of Hydrobionts Ecology and Physiology, Laboratory of Population Genetics, Akademijos-2, Vilnius-21, 08412, Lithuania. In this study the lactic acid bacteria diversity of the intestinal tract content of the vernal and autumnal migrant mallard ducks (Anas platyrhynchos) from Nemuno delta has been investigated by molecular methods: polymerase chain reaction amplification and sequencing of partial 16S rRNA genes and using culture-based techniques. The investigation of the lactic acid bacteria of the migrant mallard ducks has been performed the first time. Autumnal migrant mallard ducks in the small intestine walls (from 1.2×107 until 2.1×107 c.f.u./g) and in their content (from 3.4×107 until 1.1×108 c.f.u./g have the greatest number of the lactic acid bacteria then vernal migrants (respectively from 3.2×106 until 4.8×106 c.f.u./g and from 1.0×107 until 2.2×107 c.f.u./g). In the small intestine walls and in their content of the autumnal and vernal migrant mallard ducks, dominated cocci-shaped lactic acid bacteria (respectively 65% and 83.5%, 81.4% and 91.6%), whereas rod-shaped was under (respectively 35% and 16.5%, 18.6% and 8.4%). Supposedly, that these defferences determine some factors: a long migration, period of incubate... [to full text]

abstract: Obesity is a worldwide epidemic accompanied by multiple comorbidities. Bariatric surgery is currently the most efficient treatment for morbid obesity and its comorbidities. The etiology of obesity is unknown, although genetic, environmental, and most recently, microbiome elements have been recognized as contributors to this rising epidemic. The role of the gut microbiome in weight-loss or weight-gain warrants investigation, and bariatric surgery provides a good model to study influences of the microbiome on host metabolism. The underlying goals of my research were to analyze (i) the factors that change the microbiome after bariatric surgery, (ii) the effects of different types of bariatric surgeries on the gut microbiome and metabolism, (iii) the role of the microbiome on the success of bariatric surgery, and (iv) temporal and spatial changes of the microbiome after bariatric surgery. Roux-en-Y gastric bypass (RYGB) rearranges the gastrointestinal tract and reduces gastric acid secretions. Therefore, pH could be one of the factors that change microbiome after RYGB. Using mixed-cultures and co-cultures of species enriched after RYGB, I showed that as small as 0.5 units higher gut pH can aid in the survival of acid-sensitive microorganisms after RYGB and alter gut microbiome function towards the production of weight loss-associated metabolites. By comparing microbiome after two different bariatric surgeries, RYGB and laparoscopic adjustable gastric banding (LAGB), I revealed that gut microbiome structure and metabolism after RYGB are remarkably different than LAGB, and LAGB change microbiome minimally. Given the distinct RYGB alterations to the microbiome, I examined the contribution of the microbiome to weight loss. Analyses revealed that Fusobacterium might lessen the success of RYGB by producing putrescine, which may enhance weight-gain and could serve as biomarker for unsuccessful RYGB. Finally, I showed that RYGB alters the luminal and the mucosal microbiome. Changes in gut microbial metabolic products occur in the short-term and persist over the long-term. Overall, the work in this dissertation provides insight into how the gut microbiome structure and function is altered after bariatric surgery, and how these changes potentially affect the host metabolism. These findings will be helpful in subsequent development of microbiome-based therapeutics to treat obesity. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2016

O parvovírus humano B19 é um vírus não envelopado, que contém uma fita simples de DNA. Seu genoma é altamente conservado, com 98 a 99% de similaridade entre os isolados. Durante a última década, variantes do parvovírus B19, gênero Erythrovirus, foram descritas apresentando variabilidade genética de 11 a 14. Uma nova classificação foi proposta, dividindo o gênero em três diferentes genótipos (1, 2, 3a e 3b). Para avaliar a diversidade genética e o papel epidemiológico dos eritrovírus, 892 amostras brasileiras e chilenas, foram investigadas para a presença de DNA viral. As amostras positivas foram seqüênciadas e genotipadas através da analise da região VP1/VP2. Os resultados mostraram predominância do genótipo 1 (89 amostras), seguido do genótipo 3 (1 amostra), nas amostras brasileiras, enquanto que nas amostras chilenas, apenas o genótipo 1 foi observado (24 amostras). A única variante detectada, a amostra BR543, apresentou variabilidade de aproximadamente 13%, quando comparada ao B19, sendo classificada como genótipo 3, subtipo 3b. / Human Parvovirus B19 is a non-enveloped, ssDNA virus. The genome is highly conserved, showing 98 to 99% of nucleotide similarity between the isolates. During the last decade, parvovirus B19 variants, genus Erythrovirus, were described showing 11 to 14 %. A new classification was suggested, dividing the genus in three different genotypes (1, 2, 3a and 3b). To evaluate the epidemiology and the erythrovirus genetic diversity, 892 brazilians and chileans samples, were investigated for the virus DNA presence. The positive samples were sequenced and genotyped (VP1/VP2). The results showed the prevalence of genotype 1 (69 samples), followed by genotype 3 (1 sample), in the brazilians samples, and in the Chilean samples only genotype 1 was observed (24 samples). The variant detected, BR543, showed 13% of divergence when compared to B19, classified as genotype 3, subtype 3b.

A simbiose desenvolvida entre seres vivos e microrganismos desempenha um importante papel na relação saúde-doença do hospedeiro. Neste sentido, o corpo humano abriga uma grande e diversa comunidade de microrganismos, sendo as mucosas vaginal, intestinal e oral as principais superfícies mucosas do corpo feminino que abrigam as comunidades bacterianas de fundamental importância para a mulher. Estes microrganismos atuam no desenvolvimento e modulação do sistema imune, na manutenção e otimização de vias metabólicas e competem por sítios de colonização, prevenindo que microrganismos patogênicos estabeleçam colonização. A composição da microbiota feminina varia com a idade, pH, secreção hormonal, ciclo menstrual, uso de anticoncepcional e atividade sexual. O presente estudo buscou caracterizar a composição da microbiota do corpo feminino durante o período gestacional, comparando os achados entre gestantes e não gestantes saudáveis, através de técnicas de biologia molecular. Foram selecionadas 60 mulheres saudáveis para o estudo e coletadas amostras de secreção vaginal, fezes e swab oral de cada participante. O DNA das amostras foi extraído e submetido à sequenciamento do gene 16S rRNA e quantificado através da técnica de PCR em tempo real. Das participantes selecionadas, 42 eram gestantes e 18 eram mulheres não gestantes em idade reprodutiva. Observamos que a quantificação total de bactérias na vagina não apresentou diferenças entre gestantes e não gestantes. Houve aumento na abundância de Lactobacillus no sítio vaginal, bactérias produtoras de butirato na microbiota intestinal e Streptococcus na microbiota oral de mulheres grávidas quando comparadas com mulheres não gestantes. Além disso, observamos que a composição e a disposição dos gêneros encontrados sofrem uma modificação, tal como aumento de gêneros relacionados com a manutenção da homeostase no grupo de mulheres gestantes. O período gestacional influencia positivamente na composição da microbiota, garantindo assim a prevalência de gêneros bacterianos responsáveis pela manutenção das condições ideais para o desenvolvimento da gestação saudável. / The symbiosis developed between living organisms and microorganisms plays an important role in the health-disease relationship of the host. In this sense, the human body harbor a large and diverse community of microorganisms, the vaginal, intestinal and oral mucosa are the main mucosal surfaces of the female body that harbor bacterial communities of fundamental importance for women. These microorganisms act in the development and modulation of the immune system, in the maintenance and optimization of metabolic pathways and compete for colonization sites, preventing pathogenic microorganisms from establishing colonization. The composition of the female microbiota varies with age, pH, hormonal secretion, menstrual cycle, contraceptive use and sexual activity. The present study aimed to characterize the microbiota composition of the female body during the gestational period, comparing the findings between healthy and non - pregnant women through molecular biology techniques. Sixty healthy women were selected for the study and samples of vaginal secretion, stool and oral swab from each participant were collected. The DNA of the samples was extracted and submitted to the 16S rRNA gene sequencing and quantified by the real-time PCR technique. Were select, 42 were pregnant and 18 were non-pregnant women of reproductive age. We observed that the total quantification of bacteria in the vaginal samples did not present differences between pregnant and non-pregnant women. There was an increase in the abundance of Lactobacillus in the vaginal site, butyrate producing bacteria in the intestinal microbiota and Streptococcus in the oral microbiota of pregnant women when compared to nonpregnant women. In addition, we observed that the composition and arrangement of the genera found undergo a modification, such as an increase in genera related to the maintenance of homeostasis in the group of pregnant women. The pregnancy influences the composition of the microbiota, thus ensuring the prevalence of bacterial genera responsible for the maintenance of the ideal conditions for the development of healthy pregnancy.

Dados da Organização Mundial da Saúde (OMS) mostram que a raiva é um problema de saúde pública podendo acarretar sérios prejuízos ambientais e econômicos, a despeito da existência de vacinas eficazes de uso humano e veterinário. Segundo seu último informe, estima-se que no mundo em torno de 55.000 pessoas por ano morrem de raiva. O cão permanece como principal transmissor da raiva para o homem e também como principal vítima da doença. Nos países que conseguiram controlar a raiva em animais domésticos, o vírus se mantém circulante na natureza por meio dos animais silvestres, sendo os morcegos apontados como a segunda espécie transmissora da raiva a humanos. Os Lyssavirus têm sido detectados em morcegos, em diversos continentes, sendo identificados como transmissor em dez das onze espécies de Lyssavirus. Fósseis de morcego mostram sua presença há 50 milhões de anos. Mas somente em 1911, Carini relacionou pela primeira vez a raiva aos morcegos, levantando a hipótese destes serem os transmissores da doença a outros animais. Há registros de que o vírus da raiva foi isolado em pelo menos 41 das 167 espécies de morcegos brasileiras, sendo que a maioria dessas espécies está relacionada a atividades humanas com a presença destes animais próximos ao local de trabalho e moradia das pessoas. Os morcegos hematófagos Desmodus rotundus são encontrados do norte do México até a costa norte do Chile, região central da Argentina e costa do Uruguai e com exceção do Chile. Esta espécie de morcego tem sido apontada como reservatório natural do vírus da raiva nesta região. Alguns pesquisadores observaram que a raiva em morcegos não hematófagos precede a raiva bovina e em animais de estimação, sugerindo que os morcegos não hematófagos podem ser o elo entre a raiva silvestre e a raiva urbana e o fato de se detectar a variante mantida por morcegos hematófagos Desmodus rotundus em cães e gatos mostra que o papel deste morcego no ciclo da raiva não está limitado à raiva silvestre. As características dos Lyssavirus adaptados a morcegos têm mostrado diferenças quando comparadas à raiva relacionada aos carnívoros, confirmando a necessidade do desenvolvimento de metodologias que permitam estudos complementares mais precisos a respeito da biologia e epidemiologia da raiva em quirópteros. A escassez de dados na literatura, até o momento, a respeito do genoma completo da variante do vírus da raiva mantida por populações de morcegos hematófagos Desmodus rotundus, deixa uma lacuna no entendimento da epidemiologia molecular deste vírus. A importância epidemiológica desta espécie na transmissão da raiva é inquestionável. Neste estudo foi sequenciado e analisado, o genoma da variante do vírus da raiva mantido por populações de morcego hematófago Desmodus rotundus isolado de um morcego hematófago Desmodus rotundus. A amostra, procedente de área endêmica no Estado de São Paulo, foi filogeneticamente comparada com o genoma da amostra padrão para a espécie viral 1 - Rabies virus e outras amostras pertencentes ao ciclo aéreo ou terrestre de transmissão, disponíveis no GenBank, identificando possíveis padrões de diferenciação, próprios do ciclo aéreo, e em alguns casos relacionados somente à variante estudada. / Data from the World Health Organization (WHO) show that rabies is a public health problem which can cause serious environmental and economic damage, despite the existence of effective vaccines for human and veterinary use. According to WHO latest report, estimated that worldwide around 55,000 people per year died of rabies. The dog remains the main transmitter of rabies to humans as well as the main victim of the disease. In countries that were successful in controlling rabies in domestic animals, the virus is still circulating in nature by wild animals and the bats are seen as the second species transmitting rabies to humans. The Lyssavirus have been detected in bats in several continents and is identified as a transmitter in ten of eleven species of Lyssavirus. Bat fossils show their presence for 50 million years. But only in 1911, in the first time Carini related to rabies at bats, raising the possibility of these being the transmitters of the disease to other animals. Reports show that the Rabies virus was isolated in at least 41 of the 167 species of bats in Brazil, with the majority of these species is related to human activities with the animals living near the local job and houses of people. The vampire bat Desmodus rotundus is found from northern Mexico to northern Chile coast, central coast of Argentina and Uruguay and with the exception of Chile. This bat species has been identified as a natural reservoir of the Rabies virus in this region. Some researchers observed that rabies into non-hematophagous bats precedes the bovine rabies and in pets, suggesting that the non-hematophagous bats may be the link between wildlife rabies and urban rabies and the fact that detect the variant maintained by vampire bats Desmodus rotundus in dogs and cats shows that the role of bat rabies in the cycle is not limited to wildlife rabies. The characteristics of Lyssavirus bat adapted have been shown differences when compared to rabies related to the carnivores, confirming the need to develop methods that enable more accurate follow-up studies about the biology and epidemiology of rabies in bats. The paucity of data in the literature to date about the complete genome of the Rabies virus variant maintained by populations of vampire bats Desmodus rotundus leaves a gap in understanding the molecular epidemiology of this virus and the epidemiological importance of this species in the transmission of Rabies virus is unquestionable. In this study we sequenced and analyzed the genome of the Rabies virus variant maintained by populations of bat Desmodus rotundus isolated from a bat Desmodus rotundus. The sample, coming from an endemic area in São Paulo, was phylogenetically compared with the genome of the standard sample for spcies 1 - Rabies virus and other samples belonging to the Terrestrial and Aerial cycles of transmission, available in GenBank, to identify possible patterns of differentiating themselves Aerial cycle and in some cases linked only to variant studied.

A mastite é a inflamação da glândula mamária cuja causa mais comum são as infecções bacterianas. É considerada a doença de maiores prejuízos e prevalência em vacas leiteiras. A forma subclínica da mastite é o tipo mais predominante de infecção intramamária (IIM), sendo Corynebacterium spp. um dos agentes mais frequentemente isolados. Portanto, os objetivos gerais deste estudo foram: 1) avaliar o efeito de IIMs subclínicas causadas por Corynebacterium spp. sobre a composição e a produção de leite de quartos mamários; 2) determinar o efeito da IIM causada por Corynebacterium spp. sobre o teor de gordura, proteína bruta, lactose, caseína, extrato seco total, extrato seco desengordurado do leite e contagem de células somáticas (CCS); 3) avaliar a técnica de Espectrometria de massas por Ionização e Dessorção à Laser Assistida por Matriz Tempo de Voo (MALDI/TOF-MS) para a identificação das espécies de Corynebacterium isoladas de quartos mamários. Foram utilizados 21 rebanhos leiteiros (n=1.242 animais), nos quais foram selecionadas vacas em lactação previamente identificadas com IIM causada por Corynebacterium spp. (n=285). Amostras de leite de quartos mamários de vacas previamente identificadas com IIM causada por Corynebacterium spp. (n=1.140) foram coletadas, identificadas por meio de cultura microbiológica e criopreservadas. Os isolados de Corynebacterium spp. (n=351) foram descongelados, recultivados e submetidos à identificação pela técnica de sequenciamento de genes 16S rRNA (standard gold) e MALDI/TOF-MS. Para a análise de MALDI/TOF-MS, os isolados bacterianos foram submetidos a protocolo de extração em tubo, baseado no uso de ácido fórmico. Do total de quartos mamários infectados por Corynebacterium spp. (n=190) identificados pelo sequenciamento de genes 16S rRNA, 46 quartos mamários apresentaram contralaterais sadios (controle). Para a coleta de amostras de leite para composição e CCS, as vacas foram submetidas à ordenha completa e individual dos quartos mamários. Foi avaliado o efeito de IIM causada por Corynebacterium spp. sobre a produção e a composição do leite, utilizando delineamento em parcelas subdivididas em faixas (strip-plot). Todos os quartos mamários infectados por Corynebacterium spp. (n=190) apresentaram média de CCS de 690x103células/mL. Dentre os quartos contralaterais, a CCS foi maior para os quartos com IIM causada por Corynebacterium spp. (194x103células/mL) que em quartos não infectados (81x103células/mL). Não houve efeito de Corynebacterium spp. sobre a produção de leite, gordura, proteína, caseína, lactose, sólidos totais e extrato seco desengordurado. Um total de 222 isolados foram identificados pela MALDI/TOF-MS como Corynebacterium spp., dos quais, Corynebacetium bovis foi o mais frequentemente isolado (n = 208). Espécies não-lipofíficas representaram 4,5% do total de isolados (n=10), destes C. auriscanis (n=3), C. xerosis (n=3), C. amycolatum (n=1), C. casei (n=1), C. efficiens (n=1), C. pseudotuberculosis (n=1). Apenas quatro amostras (1,8%) não foram identificadas ao nível de espécie, Corynebacterium spp. Houve equivalência de resultados de 95% entre as duas técnicas de identificação utilizadas. Em conclusão, IIMs causadas por Corynebacterium spp. não apresentam efeito sobre a produção e composição de leite, porém apresentam aumento significativo da CCS. Adicionalmente, a técnica de MALDI/TOF-MS com o uso da extração em tubo por ácido fórmico/acetonitrila foi aplicada com 91,57% de sensibilidade de identificação das espécies de Corynebacterium nos casos de mastite bovina subclínica, quando comparado aos resultados do sequenciamento de genes 16S rRNA. / Mastitis is an inflammation of the mammary glands which bacteria are the most common cause of infections. It is considered a disease with higher prevalence and losses in dairy cows. The subclinical form of mastitis is the most prevalent type of intramammary infection (IMI), and Corynebacterium spp., one of the agents most frequently isolated. Therefore, the main objectives of this study were: 1) to evaluate the effect of subclinical IMI caused by Corynebacterium spp. on milk composition and yield of mammary quarters; 2) to determine the effect of IMI caused by Corynebacterium spp. on the concentration of fat, protein, lactose, casein, total solids, non fat dry milk and somatic cell count (SCC); 3) to evaluate the technique of Matrix-Assisted Laser Desorption Ionization - Time-of-Flight - Mass Spectrometry (MALDI/TOF-MS) for species identification of Corynebacterium isolated from mammary glands. Twenty-one dairy herds were used (n = 1242 animals) on which lactating cows were selected previously identified with IMI caused by Corynebacterium spp. (n = 285). Samples of milk from mammary glands of cows previously identified with IMI caused by Corynebacterium spp. (n = 1140) were collected, identified by microbiological culture and cryopreserved. The strains of Corynebacterium spp. (n = 351) were thawed and recultivated wich were submitted for identification by the technique of sequencing 16S rRNA (gold standard) and MALDI/TOF-MS. For analysis of MALDI/TOF-MS, the isolates were subjected to tube-extraction protocol based on the use of formic acid and acetonitrile. Of the total number of mammary quarters infected by Corynebacterium spp. (n = 190) identified by sequencing 16S rRNA genes, 46 showed contralateral healthy mammary quarters (control). For the collection of milk composition samples and SCC, cows were submitted to a complete milking in individual mammary quarters. The effect of IMI caused by Corynebacterium spp. on milk yield and composition were analyzed using strip-plot design. All mammary quarters infected with Corynebacterium spp. (n = 190) had mean of CCS 690x103cells/mL. Among the contralateral quarters, CCS was higher in quarters with IMI caused by Corynebacterium spp. (194x103células/mL) comparing with uninfected quarters (81x103cells/mL). There was no effect of Corynebacterium spp. on milk yield, on the concentration of fat, protein, casein, lactose, total solids and solids not fat. A total of 222 isolates were identified by MALDI/TOFMS as Corynebacterium spp. of which, Corynebacetium bovis were the most isolated (n = 208). Species no-lipophilic represented 4.5% of isolates (n = 10) of these C. auriscanis (n = 3), C. xerosis (n = 3), C. amycolatum (n = 1), C. casei (n = 1), C. efficiens (n = 1), C. pseudotuberculosis (n = 1). Only four samples (1.8%) were not identified to the specie level. There were equivalence results of 95% between the two identification techniques used. In conclusion, IMI caused by Corynebacterium spp. have no effect on milk yield and composition, but exhibit a significant increase in CCS. Additionally, the technique of MALDI/TOF-MS using the tube-extraction by formic acid/acetonitrile was applied with 91.57% sensitivity for identifying the species of Corynebacterium on cases of bovine mastitis subclinical when compared to the results of genes sequencing 16S rRNA.

Contamination of groundwater that is suitable for drinking is of growing concern as the water supply of South Africa is becomingincreasingly limited. This is especially the case in the North West province, with its semi – arid climate and variable rainfall patterns. The aim of the study was to evaluate the microbiological and physico – chemical qualities of groundwater obtained from selected DWA (Department of Water Affairs) monitoring boreholes in the Mooi River and Harts River catchment areas. Physico -chemical parameters included temperature, pH, electrical conductivity (EC), salinity, total dissolved solids (TDS), sulphate and nitrate concentrations. Physical parameters were measured using a calibrated submerge-able multimeter and chemical parameters using specialised kits and a spectrophotometer. Microbiological parameters included heterotrophic plate counts and total and faecal coliform enumeration. Membrane filtration and culture based methods were followed for enumeration of bacteria. During the identification procedures multiplex PCR for E. coli identification and 16S rRNA gene sequencing for identification of heterotrophic plate count bacteria and amoeba resistant bacteria were used. For antibiotic resistance, the Kirby- Bauer (1996) disk diffusion method was used. During the warm and wet season high electrical conductivity and salinity were observed in the Trimpark (65.3 mS/m; 325 ppm), School (125.1 mS/m; 644 ppm), Warrenton (166.9 mS/m; 867 ppm) and Ganspan (83.3 mS/m; 421 ppm) boreholes. Warrenton borehole had a high sulphate level (450 mg/l) as well. High chemical oxygen demand was observed in the Blaauwbank (62 mg/l) and Warrenton (98.5 mg/l) boreholes. In the dry and cold season similar observations were made for the various boreholes. Electrical conductivity and salinity levels remained high for the Trimpark (70.1 mS/m; 427.5 ppm), School (127 mS/m; 645 ppm), Warrenton (173.3 mS/m; 896.5 ppm) and Ganspan (88.1 mS/m; 444.5 ppm) boreholes. Nitrate levels for the Trimpark (14.1 mg/l) and School (137 mg/l), as well as sulphate levels for the Warrenton (325 mg/l) borehole were also high. Total coliforms, faecal streptococci and HPC bacteria were enumerated from water samples from all boreholes, except Blaauwbank where no faecal streptococci were enumerated. Faecal coliforms were enumerated from 5 of the possible 7 boreholes during a warm and wet season (Trimpark – 42 cfu/100ml; School – 2 cfu/100ml; Cemetery – 175 cfu/100ml; Warrenton – 3.84 x 10³ cfu/100ml; Ganspan – 1.9 x 10³ cfu/100ml). Indicator bacteria (FC, TC, HPC) exceeded target water quality ranges (TWQR) for drinking water in each case. During the cold and dry sampling season, faecal coliforms were enumerated mainly from the Trimpark (11 cfu/100ml) borehole. Total coliforms, faecal streptococci and HPC bacteria were enumerated from all the boreholes, except for Blaauwbank that contained no faecal streptococci or total coliforms. Enumerated indicator bacteria levels again exceeded TWQR for domestic use. Total coliform counts for the Pad dam borehole, however, complied with TWQR for domestic use. Identified E. coli were resistant to Erythromycin, Cephalothin and Amoxicillin and susceptible to Ciprofloxacin. Escherichia coli isolated from the Mooi River catchment shared the same antibiotic resistance phenotype. The most abundant HPC bacterial genus identified was Pseudomonas spp. (7 isolates). Opportunistic pathogens isolated included Pseudomonas aeruginosa, Acinetobacter, Aeromonas, Alcaligenes, Flavobacterium, Bacillus cereus and Mycobacterium spp. Varying degrees of antibiotic resistance were observed. Generally, the same pattern between the same genera were observed. All HPC isolates were resistant to Cephalothin and Amoxicillin and a lower degree Erythromycin and Streptomycin. The most abundant amoeba resistant bacteria was identified as Pseudomonas spp. Other isolates included Alcaligenes faecalis and Ochrobactrum sp. and Achromobacter sp. All of these are opportunistic pathogens, except for Achromobacter. Resistance to more antibiotics (Streptomycin, Chloramphenicol, Cephalothin, and Amoxicillin) was observed in ARBs compared to HPC (Cephalothin, Amoxicillin) from bulk water from the same borehole. The water of all the aquifers sampled is of very poor physico - chemical or microbiological quality or both. Water may be used for irrigation or livestock watering only in the case where these boreholes comply with TWQR for said purposes. Results obtained indicate that the groundwater is faecally contaminated. Amongst the bacteria, opportunistic pathogens displaying various degrees of antibiotic resistance were frequently isolated. These results indicate health risks if untreated groundwater is consumed. Therefore groundwater needs to be treated before distribution especially if the water is for human consumption. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2013.