Thirteen cycling, nonlactating dairy cows were hormonally treated to induce lactation. Starting 7 days after estrus and continuing for 7 days progesterone and estradiol-17β dissolved in absolute ethanol were administered twice daily. Daily dose level for progesterone and estradiol- 178 was .25 mg and .01 mg/kg body weight, respectively. Intramuscular injections of dexamethasone (.028 mg/kg body weight) were given on days 18, 19 and 20 after initial treatment. Mammary tissue biopsies were taken from 9 cows on days 18, 21 and 23 after initiation of treatment (3 cows each day). Histological analysis was performed (> 30,000 contacts) to determine the average percent tissue area composed of epithelium, stroma, lumen, adipose and duct. Difference in percent area between days 18 thru 23 was as follows: (1) epithelium +11.1%, (2) stroma -25.9%, (3) lumen +13.9%, (4) adipose +1.58 and (5) duct -.74%. Also, an increase of 15 nuclei/alveoli occurred between days 18 thru 23. On day 18 small lipid droplet epithelia predominated but was replaced with mixed lipid droplet epithelia by day 23. Day 18 epithelia had elongated dark staining nuclei. Epithelia on day 23 had spherically shaped nuclei containing several nucleoli and a decreased nuclear/cytoplasmic ratio when compared to day 18 epithelia. Four of the hormonally treated cows were allowed to lactate. Production from these four cows ranged from 33 to 74% of the previous lactation. Production and histological development were variable in cows treated with estrogen, progesterone and dexamethasone. / Master of Science

A study was conducted to examine the correlation between a novel behavior monitoring system and a validated data logger. We concluded that the behavior monitoring system was valid for tracking daily rest time in dairy cows (R=0.96); however the correlation values for rest bouts and rest duration were relatively low, (R=0.64) (R=0.47), respectively. Daily monitoring of animal activity and milk components can be used to detect mastitis prior to clinical onset. Data from 268 cases with clinical mastitis and respective controls (n=268) from Virginia Tech and the University of Florida dairy herds were examined. Variables collected included daily milk yield, electrical conductivity, milk fat, protein, and lactose percent, as well as activity measurements including daily rest time, daily rest duration, daily rest bouts, and daily steps taken. Variables were collected for case and control cows in the 14 d prior to and after clinical diagnosis, for a total 29 d monitoring period. A milk sample was aseptically collected upon detection of clinical signs as observed by milker's at both farms. A statistical method (candisc discriminant analysis) was used to combine all measurements and sensitivity and specificity was calculated. Virginia Tech cows on d -1 (sensitivity=95%, specificity=95%), Virginia Tech and University of Florida cows on d -1 (sensitivity=88%, specificity=90). Overall, daily monitoring of animal activity and milk components can detect mastitis prior to onset of clinical signs of disease. This may allow producers to intervene and make proactive management decisions regarding herd health prior to clinical diagnosis. / Master of Science

This study investigated the hypothesis that the particulate content of milk, as monitored with particle counters, is correlated to the health status of lactating dairy cows, in particular the condition mastitis. Twenty Holstein cows were monitored from the very first day of clinical mastitis outbreak until complete recovery from the disease. During the experiment, the changes in particle behaviour in all four quarters and mixture of milk from all of them were measured. For each sample the following parameters were measured: somatic cell count, fat content, lactose and protein concentration, number and size distribution of milk particles, electric conductivity and diameter of milk fat globules. In total over thirty mastitis outbreaks were observed and monitored throughout, including the first phase of this study when over three thousand samples of foremilk were collected and examined. An operational protocol and particle monitoring device were designed with the help of a commercial company Facility Monitoring Systems Ltd (FMS), Malvern. A particle counter and Peak Height Analyser (PHA) were used to monitor particulate content of milk and a compound phase contrast microscope was used to identify milk particles by photographic visualisation and to establish their diameter. It was observed that the number of particles, milk fat globule diameter and somatic cell counts were stable during periods without udder inflammation. Mastitis caused great changes in these parameters. Both milk particulate size and number were significantly affected by clinical and subclinical form of inflammation (change to the particulate behaviour). It was observed that the changes to the volume median diameter (VMD) of fat globules became evident a few days before clinical signs were present. Results obtained from a particle counter and the PHA were in agreement with data obtained by microscopy. Major changes were recorded in the number of total particles in milk before and during the outbreak of mastitis. Further research showed that changes took place in the pattern of particulate behaviour without visible signs of disease; additional data established that subclinical mastitis can be also identified through the monitoring of particles in milk. In summary monitoring of the behaviour (changes to size and number) of milk fag globules (MFG) can be used as an early indicator of the onset of mastitis. In addition data collected during study produced strong evidence supporting the theory of the interdependence of the quarters within the udder. It was found that the coefficient of correlation for size and number of particles for all four quarters within the udder was statistically significant. Particle counts and the VMD values behaviour were similar for the four quarters. This relationship was observed for all monitored animals. Moreover, the same relationship was also observed during both clinical and subclinical outbreaks of mastitis. Somatic cell count was affected only in an infected quarter while particulate content of milk ―responded‖ to disease in all four quarters within the udder (even if only one was infected). These results were the most surprising and unexpected outcome, suggesting that four quarters within the udder work together as one organ not four separate units. It was observed that the mean MFG cannot be used as a baseline to test individual animal deviations due to the unique particle profile of each observed animal. In all monitored animals particle counts obtained from PHA was found to be in the range of 1011 to 1013 with an average of 1012 particles per ml. The number of particles recorded in mastitis for one animal was at the healthy level for another. The particle pattern became a finger print for each animal and therefore MFG behaviour cannot be compared between animals. Following the first phase of the study the monitoring period was set at 15 to 20 days. This protocol allowed for minimising the influence of any other parameters on particles, which may influence the outcome of an experiment e.g. the number of particles in single samples detected by the particle counter. It was also essential to understand how the age, nutrition and stage of lactation might influence the particles and affect the results. Therefore two animals were chosen to be examined during their lactation. The analysed data did not present enough evidence to establish the relationship between nutrition and milk fat globules size and number. However to better understand this association additional studies should be carried out. Further work is required to optimise the monitoring device and build a fully automated system which will allow collecting and analysing data from the whole herd. This study proposed that particle pattern is unique for each animal – like a finger print. More research is needed to better understand the mechanism behind milk fat globules synthesis during inflammation. The results obtained during this study provide new evidence with regard to physiological changes within the udder before and during mastitis outbreak and supported the theory of interdependence between quarters within udder. The particle count in milk can be used as an indicator of the health status of the single animal. Combined the PHA and microscope can be used as a new tool to determine and monitor the particle count in milk. The understanding of the particulate behaviour will help to minimise the chance of mastitis outbreak by early detection and also to reduce the chance of the cross-contamination between animals during the milking process. Milk fat globule size and number can be used as an efficient indicator of the onset of mastitis.

Atualmente a terapia e as práticas de manejo da mastite estão baseadas na administração de agentes antimicrobianos com ampla atividade contra os microrganismos mais comuns desta doença. Os fármacos são administrados diretamente no canal da teta e/ou por sistêmica. Os tratamentos com antimicrobianos são realizados durante o período seco como terapia preventiva, ou durante o período de lactação como terapia curativa. O leite produzido por animais tratados pode conter resíduos do medicamento. A presença de resíduos de antimicrobianos no leite constitui um risco para a saúde pública devido ao fenômeno de múltipla resistência, bem como uma causa de perdas econômicas, considerando a fabricação de queijo e iogurte, entre outros derivados lácteos. A proposta deste estudo foi avaliar alguns fatores que contribuem para a ocorrência de resíduos em vacas tratadas além do período recomendado para descarte. Os fatores avaliados foram: fisiológico, como a produção leiteira; clínico, como a mastite e farmacológico como a via de administração e o fármaco utilizado. Foi usado um teste comercial para a detecção de resíduos (Delvotest®) por inibição microbiológica. Entre os fatores estudados, foi demonstrada a influência da produção leiteira, ou seja, animais com produção leiteira maior do que 20L/dia apresentaram menor ocorrência de resíduo (39,5%), quando comparado com aqueles com produção menor do que 20L/dia (70,7%) e a diferença foi significante (P < 0,0001). Entre os fatores clínicos foi determinado que a presença e intensidade do processo inflamatório contribuem para a ocorrência de resíduos. Deste modo, entre os quartos com mastite clínica tratados o nível de resíduo foi 47,7%, os quartos com mastite subclínica tratados apresentaram 34,9% e os controles 9,5% (quartos sem mastite tratados) e a diferença entre os três grupos foi significante (P = 0,0381, P = 0,0008, P = 0,0209). Ao se comparar a ocorrência de resíduos no leite de quartos tratados durante a lactação (41,9%) com os resíduos nos quartos que receberam a terapia de vaca seca (23,2%) foi observado um maior nível entre os grupos tratados durante a lactação e a diferença foi significante (P < 0,0001). Em relação aos fatores farmacológicos, a via de aplicação bem como o grupo farmacológico do antimicrobiano utilizado no tratamento foram demonstradas diferenças estatísticas tanto para os diversos antimicrobianos, quanto para as vias de administração. Um maior nível de resíduo foi observado entre os grupos farmacológicos em quartos tratados por via intramamária com aminoglicosídeos (gentamicina) (66,7%). E o maior nível de resíduo foi observado quando ambas as vias foram utilizadas simultaneamente, tanto para aminoglicosídeos (93,7%), quanto para betalactâmicos (100%). O conhecimento de que o pKa dos antimicrobianos irá determinar diferentes níveis de distribuição nos órgãos e tecidos sustenta a hipótese que a via de administração pode influenciar a presença de resíduos de antimicrobianos no leite. Conseqüentemente, o uso simultâneo das duas vias de administração, uma prática comum, contribuiu também para um alto risco da ocorrência de resíduos de antimicrobianos. Deve-se ressaltar que a redução da presença de resíduos no leite não depende apenas de normas e regulamentos, mas envolve uma orientação aos produtores e aos veterinários para controlar e/ou prevenir os fatores de risco. Assim, os resultados deste estudo irão contribuir para esclarecer alguns aspectos importantes para um melhor entendimento da ocorrência de resíduo no leite além do período recomendado para descarte. / Current mastitis therapy and management practices are base on the administration of antimicrobial agents with high activity against most common mastitis bacterial pathogens. Drugs are administrated directly into the teat canal and/or systemically. Antibiotic treatments are performed during the dry period as preventive therapy, or during the lactating periods as therapeutic cure. Milk produced from treated animals may contain drug residues. The presence of antibiotic residues in milk constitutes a public health risk due to the multi-resistance phenomenon, as well as, a source of economical losses, considering the industries of cheese and yogurts, among others dairy products. The purpose of this study was evaluated some factors that contribute to the occurrence of residues in treated cows beyond the recommended discarded period. The evaluated factors were: physiological, as milk production, clinical, as mastitis and pharmacological, as routes and antimicrobials. It was used a commercial test for residues detection (Delvotest®) by microbiological inhibition. Among the clinical factors it was demonstrated the influence of the milk production, so animals with milk production higher than 20 L/day showed lower occurrence of residues (39.5%), compared to the ones with less than 20 L/day (70.7%) the difference was significant (P<0.0001). Among the clinical factors it was determinated that the presence of inflammatory process and the intensity of it, contributes to the presence of residues. Therefore, among the clinical mastitis treated quartes the level of residues was 47.7%, while the subclinical treated quartes showed 34.9% and, the control ones 9.5% (i.e. treated quartes without mastitis) and the difference among the three groups was significant (P=0.0381, P=0.0008, P=0.0209).Comparing the residues in milk from treated quartes during lactation (41.9%) with the quartes that received the dry cow therapy (23.2%) it was observed a higher level of residues among the groups treated during the lactation and the difference was significant (P<0.0001). Among the pharmacological factors, the routes as well the pharmacological group of the antimicrobials used in the treatment showed statistical differences either among the antimicrobials as well as the administration routes. A high level of residues was observed among pharmacological groups in quartes treated by intramammarian route with aminoglicosides (gentamicin) (66.7%). And, the highest level of residues was observed when both route where used simultaneously, either with aminoglicosides (93.7%) as well as with betalactamics (100%). The knowledge that different pKa among the antimicrobials will determine the level of distribution in different organs and tissues, support the hypothesis that the administration route could influence the presence of residues of antibiotics in milk. Consequently, the simultaneous use of both administration routes, a very usual practice, could also contributes to a higher risk of antimicrobial residue occurrence. To reduce this possibility, it is not only a matter of Regulatory Affairs, but also involves the dairy farmers and veterinarians\' proper orientation about how to avoid and to control the risk factors, therefore, the results of this study will contribute to clarify some important aspects to a better understanding of the occurrence of residues in milk beyond the recommend discard period.

Atualmente a terapia e as práticas de manejo da mastite estão baseadas na administração de agentes antimicrobianos com ampla atividade contra os microrganismos mais comuns desta doença. Os fármacos são administrados diretamente no canal da teta e/ou por sistêmica. Os tratamentos com antimicrobianos são realizados durante o período seco como terapia preventiva, ou durante o período de lactação como terapia curativa. O leite produzido por animais tratados pode conter resíduos do medicamento. A presença de resíduos de antimicrobianos no leite constitui um risco para a saúde pública devido ao fenômeno de múltipla resistência, bem como uma causa de perdas econômicas, considerando a fabricação de queijo e iogurte, entre outros derivados lácteos. A proposta deste estudo foi avaliar alguns fatores que contribuem para a ocorrência de resíduos em vacas tratadas além do período recomendado para descarte. Os fatores avaliados foram: fisiológico, como a produção leiteira; clínico, como a mastite e farmacológico como a via de administração e o fármaco utilizado. Foi usado um teste comercial para a detecção de resíduos (Delvotest®) por inibição microbiológica. Entre os fatores estudados, foi demonstrada a influência da produção leiteira, ou seja, animais com produção leiteira maior do que 20L/dia apresentaram menor ocorrência de resíduo (39,5%), quando comparado com aqueles com produção menor do que 20L/dia (70,7%) e a diferença foi significante (P < 0,0001). Entre os fatores clínicos foi determinado que a presença e intensidade do processo inflamatório contribuem para a ocorrência de resíduos. Deste modo, entre os quartos com mastite clínica tratados o nível de resíduo foi 47,7%, os quartos com mastite subclínica tratados apresentaram 34,9% e os controles 9,5% (quartos sem mastite tratados) e a diferença entre os três grupos foi significante (P = 0,0381, P = 0,0008, P = 0,0209). Ao se comparar a ocorrência de resíduos no leite de quartos tratados durante a lactação (41,9%) com os resíduos nos quartos que receberam a terapia de vaca seca (23,2%) foi observado um maior nível entre os grupos tratados durante a lactação e a diferença foi significante (P < 0,0001). Em relação aos fatores farmacológicos, a via de aplicação bem como o grupo farmacológico do antimicrobiano utilizado no tratamento foram demonstradas diferenças estatísticas tanto para os diversos antimicrobianos, quanto para as vias de administração. Um maior nível de resíduo foi observado entre os grupos farmacológicos em quartos tratados por via intramamária com aminoglicosídeos (gentamicina) (66,7%). E o maior nível de resíduo foi observado quando ambas as vias foram utilizadas simultaneamente, tanto para aminoglicosídeos (93,7%), quanto para betalactâmicos (100%). O conhecimento de que o pKa dos antimicrobianos irá determinar diferentes níveis de distribuição nos órgãos e tecidos sustenta a hipótese que a via de administração pode influenciar a presença de resíduos de antimicrobianos no leite. Conseqüentemente, o uso simultâneo das duas vias de administração, uma prática comum, contribuiu também para um alto risco da ocorrência de resíduos de antimicrobianos. Deve-se ressaltar que a redução da presença de resíduos no leite não depende apenas de normas e regulamentos, mas envolve uma orientação aos produtores e aos veterinários para controlar e/ou prevenir os fatores de risco. Assim, os resultados deste estudo irão contribuir para esclarecer alguns aspectos importantes para um melhor entendimento da ocorrência de resíduo no leite além do período recomendado para descarte. / Current mastitis therapy and management practices are base on the administration of antimicrobial agents with high activity against most common mastitis bacterial pathogens. Drugs are administrated directly into the teat canal and/or systemically. Antibiotic treatments are performed during the dry period as preventive therapy, or during the lactating periods as therapeutic cure. Milk produced from treated animals may contain drug residues. The presence of antibiotic residues in milk constitutes a public health risk due to the multi-resistance phenomenon, as well as, a source of economical losses, considering the industries of cheese and yogurts, among others dairy products. The purpose of this study was evaluated some factors that contribute to the occurrence of residues in treated cows beyond the recommended discarded period. The evaluated factors were: physiological, as milk production, clinical, as mastitis and pharmacological, as routes and antimicrobials. It was used a commercial test for residues detection (Delvotest®) by microbiological inhibition. Among the clinical factors it was demonstrated the influence of the milk production, so animals with milk production higher than 20 L/day showed lower occurrence of residues (39.5%), compared to the ones with less than 20 L/day (70.7%) the difference was significant (P<0.0001). Among the clinical factors it was determinated that the presence of inflammatory process and the intensity of it, contributes to the presence of residues. Therefore, among the clinical mastitis treated quartes the level of residues was 47.7%, while the subclinical treated quartes showed 34.9% and, the control ones 9.5% (i.e. treated quartes without mastitis) and the difference among the three groups was significant (P=0.0381, P=0.0008, P=0.0209).Comparing the residues in milk from treated quartes during lactation (41.9%) with the quartes that received the dry cow therapy (23.2%) it was observed a higher level of residues among the groups treated during the lactation and the difference was significant (P<0.0001). Among the pharmacological factors, the routes as well the pharmacological group of the antimicrobials used in the treatment showed statistical differences either among the antimicrobials as well as the administration routes. A high level of residues was observed among pharmacological groups in quartes treated by intramammarian route with aminoglicosides (gentamicin) (66.7%). And, the highest level of residues was observed when both route where used simultaneously, either with aminoglicosides (93.7%) as well as with betalactamics (100%). The knowledge that different pKa among the antimicrobials will determine the level of distribution in different organs and tissues, support the hypothesis that the administration route could influence the presence of residues of antibiotics in milk. Consequently, the simultaneous use of both administration routes, a very usual practice, could also contributes to a higher risk of antimicrobial residue occurrence. To reduce this possibility, it is not only a matter of Regulatory Affairs, but also involves the dairy farmers and veterinarians\' proper orientation about how to avoid and to control the risk factors, therefore, the results of this study will contribute to clarify some important aspects to a better understanding of the occurrence of residues in milk beyond the recommend discard period.

Objetivou-se neste trabalho, comparar se o tratamento homeopático eliminou ou não o agente causal das mastites subclinicas; se houve alteração na contagem de células somáticas (CCS) e a variação da produção láctea antes e depois do tratamento homeopático proposto. Utilizou-se 34 glândulas mamárias de 22 vacas que apresentavam mastite subclinica, CMT 2+ e 3+, pluríparas entre o terceiro e sexto me de lactação e entre a segunda e a quinta cria. Foram divididas em 2 grupos. O grupo teste-homeopatia recebeu 15 glóbulos do medicamento Phytolaca decandra 6 CH duas vezes ao dia,e o grupo controle recebeu 15 glóbulos inertes, duas vezes ao dia, por 15 dias consecutivos. Ao final do experimento, não houve diferença significativa entre as contagens de células somáticas da secreção láctea das glândulas quando comparadas antes e depois do tratamento homeopático, bem como quando comparou-se ambos os grupos. Porém, observou-se que a produção láctea dos animais tratados com remédio homeopático aumentou significativamente (P<0,05) em 2,5 kg/leite/dia em média. No grupo tratado com placebo não houve diferença significativa na produção láctea no mesmo período. / Mammary gland inflammatory process has been particularly frequent and important in dairy cattle and gives a lot of damage. Infectious mastitis is the most important injury either by economic or public health points of view. Identify the aggressor microorganism and somatic cells count (SCC) in milk are important tools to avail the sanitary conditions of the herd and how victim react against the aggressor. In clinical mastitis, the signs of the injury and resulting reaction of the organism who suffer the injury are evident, clear, so are seem signs and symptoms of inflammation. Otherwise, in sub-clinical mastitis, signs and symptoms of inflammations are not seen, and to realize it there are technical methods. In this case, the option was use as sub-clinical mastitis indicator the Tamis test (black ground cup) and the CMT (California Mastitis Test). Then, was chosen 22 cows that obtained score 2+ and 3+ on CMT test of each teat, between third and sixth months milking and second and fifth birth. They were separated into two groups. Homeopathy is a kind of therapy that has been recently used in animals with such satisfactory results. The conventional drugs have restricted use on organic production and in same cases are forbidden. In the start of this investigation, before the homeopathic treatment, was collected, from the each teat that had sub-clinical mastitis (CMT 2+ and 3+), two milk samples. One of then was sent to somatic cells count (SCC) and another to the microbiological laboratory to identify the microorganism. Milk production from each cow, were under control all time. Since this point the group called Test-homeopathy received the homeopathic medication Phytolacca decandra 6 ch, 15 pills twice a day, and the group called Control, received placebo, 15 pills twice a day, for both groups. At the end of 15 days of treatment, a new double collect of milk samples from each same teats were done. The samples were sent again to the microbiological laboratory to identify the microorganism implicated and to somatic cells counter (SCC) laboratory. The weight of milk production of each cow was controlled before and after treatment of each cow from both groups. At the end of research, there were no significant rates between somatic cells counts (SCC) of the milk when compared before and after homeopathic treatment, as well as when compared among both groups. But, it was observed that milk production of cows which received homeopathic treatment had an increase statistically significant (P<0,05) on 2,5 kg of milk/ day in media, and the group which received placebo there were no significant rates in milk productions in the same time.

Staphylococcus aureus ist einer der bedeutendsten Erreger boviner Mastitiden. Die Vielgestaltigkeit der Resistenzmuster und Virulenzfaktoren seiner Stämme macht ihn zu einem Problemkeim aus therapeutischer und prophylaktischer Sicht. Seine Fähigkeit zur Bildung hitzestabiler Enterotoxine verleiht ihm lebensmittelhygienische Relevanz. Mehrfachresistente Stämme stellen gefährliche Zoonose-Erreger dar. Ziel der durchgeführten Untersuchung war es daher, Aufschluss über Resistenzdeterminanten und Virulenzfaktoren der in Thüringer Milchviehherden vorkommenden Staphylococcus aureus zu erhalten, wobei eine Microarray-gestützte Genotypisierung zum Einsatz kam. Weiterhin sollte analysiert werden, ob der Genotyp der Isolate mit dem Phänotyp korreliert. In 34 Thüringer Milchviehherden wurde der gesamte Bestand der laktierenden Kühe zweimal auf Basis von Viertelgemelksproben bakteriologisch untersucht. Die Beurteilung der Kulturen erfolgte im Nativausstrich nach 48-stündiger Bebrütung und zusätzlich nach Voranreicherung in einer Glucose-Bouillon mit anschließender 24-stündiger Bebrütung. Staphylococcus aureus-positiv waren 1902 von insgesamt 81 567 Milchproben. Aus diesen wurden 189 für die Herden repräsentative Isolate ausgewählt und mittels Microarray-Technologie umfassend charakterisiert und klassifiziert. Zudem wurde der Phänotyp der Isolate auf Äskulin- und Columbia-Blutagar erfasst und das Resistenzverhalten mittels Agardiffusionstest ermittelt. Die 189 typisierten Staphylococcus aureus konnten elf verschiedenen klonalen Komplexen (CC) zugeordnet werden. Der Großteil der Isolate (80,4 %) zählte zu CC133, CC151 und CC479. Diese Isolate besaßen mit einer Ausnahme das Leukozidin-Gen lukF-P83/lukM. Die übrigen Isolate, die negativ auf lukF-P83/lukM getestet wurden, gehörten acht vergleichsweise sporadisch vorkommenden CC (CC7, CC9, CC20, CC45, CC50, CC97, CC101, CC398) an. In nur 0,7 % der zu den drei dominanten CC zählenden Isolate war das Beta-Laktamase-Gen blaZ vorhanden, während es bei 54,1 % der sporadisch vorkommenden CC detektiert wurde. Das Methicillin-Resistenzgen mecA wurde bei lediglich vier Isolaten (2,1 %) nachgewiesen, die alle CC398 angehörten. Sie verfügten neben Resistenzen gegenüber β-Laktam-Antibiotika über eine Tetrazyklin-Resistenz. Darüber hinaus wurde in einem Isolat das Makrolid/Lincosamid/Streptogramin-Resistenz vermittelnde vgaA und in einem Isolat das Aminoglykosid-Resistenz vermittelnde aacA-aphD detektiert. Humanmedizinisch relevante Enterotoxin-, Exfoliatin- oder PVL-Gene wurden in den vier Methicillin-resistenten Staphylococcus aureus (MRSA) nicht gefunden. Im Agardiffusiontest zeigten diese Isolate eine Penicillin- und eine Tetrazyklin-Resistenz, jedoch keine Resistenz gegenüber Oxacillin, welches als MRSA-Marker gilt. Die Gene der klassischen, humanmedizinisch bedeutsamen Enterotoxine A, B und C waren bei 12,7 % der Isolate vorhanden, wohingegen die Gene von Enterotoxin D und E nicht vorkamen. Insgesamt fanden sich Enterotoxin-Gene bei 78,3 % der typisierten Staphylococcus aureus, wobei die für Enterotoxin G, I, M, N, O und U kodierenden dominierten. Phänotypisch unterschieden sich die CC bezüglich Hämolyse und Pigmentierung, wobei alle CC398-Isolate als eierschalenfarben mit doppelzoniger Hämolyse auftraten. Hämolysin-Gene besaßen alle Isolate, ein Zusammenhang zu den phänotypisch ausgeprägten Hämolysezonen bestand jedoch nicht. Die vorliegende Untersuchung zeigt, dass in Thüringer Milchviehbeständen zwei epidemiologisch unterschiedliche Varianten von Staphylococcus aureus existieren. Die in dieser Studie dominierenden, lukF-P83/lukM-positiven CC133, CC151 und CC479 verursachten einen Großteil der Infektionen und gelten als auf das Euter beschränkte Erreger. Sie können daher als „euterassoziiert“ angesehen werden. Dagegen verfügten die anderen in dieser Untersuchung detektierten, lukF-P83/lukM-negativen CC über Charakteristika „umweltassoziierter“ Keime. Sie besitzen ein breites Wirtsspektrum und treten auch außerhalb des bovinen Euters in der Umgebung der Kühe auf. Die Prüfung auf lukF-P83/lukM erwies sich als zuverlässige Methode, zwischen beiden epidemiologischen Varianten zu unterscheiden. Folglich lässt die An- oder Abwesenheit dieser Genkombination einen Rückschluss auf die in der Herde verbreiteten CC zu. Das ermöglicht die Berücksichtigung der CC-spezifischen Erreger-Eigenschaften bei der Etablierung von Sanierungsprogrammen, die somit effizient gestaltet werden können. MRSA waren in Thüringer Milchviehbeständen wenig verbreitet und nur schwach mit Resistenzdeterminanten und humanmedizinisch bedeutsamen Pathogenitätsfaktoren ausgestattet. Diese MRSA aus Rohmilchproben sind daher nicht mit multiresistenten Isolaten aus der Humanmedizin zu vergleichen. Gene für humanmedizinisch relevante Enterotoxine, für die ein Zusammenhang mit Lebensmittelintoxikationen belegt ist, wurden selten, andere Enterotoxin-Gene jedoch häufig nachgewiesen.

Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are the most prevalent pathogens to induce mastitis. The pathogenesis of infections induced by E. coli is sophisticatedly modulated by lipopolysaccharide (LPS), LPS binding protein, membrane CD14 (mCD14), and soluble CD14 (sCD14). In the first study, administration of recombinant bovine sCD14 (rbosCDl4) significantly reduced the fatality of LPS challenged mice and the severity of mouse mastitis in terms of clinical signs, bacterial load, and TNF-alpha production. Before investigating the potential of this strategy in dairy cows, endogenous sCD14 in milk was characterized. Based on the data of 396 quarters, the milk concentration of sCD14 was 6.67 +/- 0.44 mug/ml. The stages of lactation affected the concentration of sCD14 in milk, which was higher in transitional milk (0--4 days postpartum). Milk sCD14 also increased during an intramammary LPS challenge, which paralleled with SCC increase. The protective effect of sCD14 on bovine E. coli mastitis was then investigated. It was shown that rbosCDl4 sensitized the mammary gland to recruit leukocytes in response to LPS. To prove that the early recruitment of leukocytes plays a role in preventing intramammary E. coli infections, E. coli mastitis was induced in 9 dairy cows with or without 100 mug rbosCD14. Quarters challenged with E. coli plus rbosCD14 had a more rapid recruitment of neutrophils, a faster clearance of bacteria, reduced concentrations of TNF-alpha and IL-8 in milk, and reduced clinical symptoms than quarters injected with saline. / For S. aureus mastitis, a newly designed trivalent whole-cell vaccine being composed of the most dominant serotypes (T5, T8, and T336) was evaluated. The vaccine was immunized with or without either one of the two adjuvants, aluminum hydroxide (ALUM) and Freund's incomplete adjuvant (FICA). The vaccine, with or without the presence of adjuvants, increased antigen-specific IgG1, IgG2, but not IgM, in serum. However, all formulations only had limited effects on lymphocyte subsets, interferon (IFN)-gamma mRNA expression, and neutrophil phagocytosis in comparison with the control. / Taken together, the results indicated that increasing the concentration of sCD14 in milk might be a potential strategy to prevent or reduce severity of E. coli mastitis. On the other hand, both ALUM and FICA did not augment the immune responses when formulated with trivalent vaccine. A more immunostimulatory adjuvant will be required to improve the efficacy of the novel trivalent vaccine against S. aureus mastitis.

Bovine mammary glands respond to infection by foreign pathogens such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) through changes in gene expression. Monitoring the gene expression profiles will contribute to better understanding of the pathology of mastitis, and provide important selective markers for future animal breeding programs. Using cultured bovine mammary duct epithelial cells and somatic cells from infected bovine mammary glands, this study first examined the existence of Toll Like Receptors in these two systems. In cultured duct epithelial cells stimulated with E. coli LPS, both TLR 4 and 2 mRNA up regulation was detected at 2h-72h and 12h-48h respectively. For S. aureus LTA TLR 2 mRNA was up regulated at 48 and 72h whereas for TLR 4 mRNA expression up regulation was detected at 24, 48, and 72h in comparison to the Oh (p&lt;0.05). In the case of PGN, an abundant structural component of S. aureus, the expression of TLR 2 mRNA was significant (p&lt;0.05) at 72h whereas TLR 4 mRNA expression increased at 24, 48, and 72h. The expression of these receptors was also monitored in milk cells from cows infected with either E. coli or S. aureus. However, results obtained from the milk cells were inconclusive due to the high individual variability. Afterwards, differential gene expression profiles were monitored by the Differential Display Polymerase Chain Reaction technique in the cultured duct epithelial cells in response to E. coli and S. aureus structural components. A total of 6 candidate fragments were identified for E. coli LPS induction, whereas only one fragment was identified for S. aureus LTA induction. After LTA induction, a specific band was found to be up regulated and confirmed to be GCP-2, a chemokine involved in neutrophil recruitment. In contrast, PGN induction resulted in no change in GCP-2 levels. In different preparations of cultured duct epithelial cells both GCP-2 and IL-8 were confirmed by real time PCR to be up regulated by LTA with a significance of (p&lt;0.01) when compared to the control cells. In the case of the E. coli identified bands, a different approach is necessary to potentially confirm the origin of these fragments. Further large scale screening of the GCP-2 and IL-8 genes in dairy cattle is necessary to test for their potential use as targets to differentiate the mastitis resistant from the mastitis prone cows.

Bacteriophage therapy has been exploited for the control of bacterial diseases in fauna, flora and humans. However, the advent of antibiotic therapy lead to a cessation of most phage research. Recently, the problem of antibiotic resistance has rendered many commonly used antibiotics ineffective, thereby renewing interest in phage therapy as an alternative source of control. This is particularly relevant in the case of bovine mastitis, an inflammatory disease of bovine mammary glands, caused by strains such as Staphylococcus aureus subsp. aureus Rosenbach 1884. Antibiotic resistance (primarily towards penicillin and methicillin) by staphylococcal strains causing mastitis is regularly reported. Phage therapy can provide a stable, effective and affordable system of mastitis control with little to no deleterious effect on the surrounding environment or the affected animal itself. Several studies have delved into the field of biocontrol of bovine mastitis using phages. Results are variable. While some phage-based products have been commercialized for the treatment of S. aureus-associated infections in humans, no products have yet been formulated specifically for the strains responsible for bovine mastitis. If the reliability of phage therapy can be resolved, then phages may become a primary form of control for bovine mastitis and other bacterial diseases. This study investigated the presence of S. aureus and its phages in a dairy environment, as well as the lytic ability of phage isolates against antibiotic-resistant strains of mastitic S. aureus. The primary goals of the thesis were to review the available literature on bovine mastitis and its associated control, and then to link this information to the use of phages as potential control agents for the disease, to conduct in vitro bioassays on the selected phages, to conduct phage sensitivity assays to assess phage activity against different chemical and environmental stresses, to morphologically classify the selected phages using transmission electron microscopy, to characterize the phage proteins using one-dimensional electrophoresis, and lastly, to characterize phage genomes, using both electrophoresis as well as full genome sequencing. Twenty-eight phages were isolated and screened against four strains of S. aureus. Only six phages showed potential for further testing, based on their wide host range, high titres and common growth requirements. Optimal growth conditions for the host S. aureus strain was 37°C for 12hr. This allowed for optimal phage replication. At an optimal titre of between 6.2x10⁷ to 2.9x10⁸ pfu.mlˉ¹(at 10ˉ⁵ dilution of phage stock), these phages were able to reduce live bacterial cell counts by 64-95%. In addition, all six phages showed pathogenicity towards another 18 S. aureus strains that were isolated from different milk-producing regions during a farm survey. These six phages were named Sabp-P1, Sabp-P2, Sabp-P3, Sabp-P4, Sabp-P5 and Sabp-P6. Sensitivity bioassays, towards simulated environmental and formulation stresses were conducted on six identified phages. Phages Sabp-P1, Sabp-P2 and Sabp-P3 showed the most stable replication rates at increasing temperatures (45-70°C), in comparison to phages Sabp-P4, Sabp-P5 and Sabp-P6. The effect of temperature on storage of phages showed that 4ºC was the minimum temperature at which phages could be stored without a significant reduction in their lytic and replication abilities. Furthermore, all phages showed varying levels of sensitivity to chloroform exposure, with Sabp-P5 exhibiting the highest level of reduction in activity (74.23%) in comparison to the other phages. All six phages showed optimal lytic ability at pH 6.0-7.0 and reduced activity at any pH above or below pH 6.0-7.0. Exposure of phages to varying glycerol concentrations (5-100%) produced variable results. All six phages were most stable at a glycerol concentration of 10-15%. Three of the six isolated phages, Sabp-P1, Sabp-P2 and Sabp-P3, performed optimally during the in vitro assays and were used for the remainder of the study. Morphological classification of phages Sabp-P1, Sabp-P2 and Sabp-P3 was carried out using transmission electron microscopy. All three phages appeared structurally similar. Each possessed an icosahedral head separated from a striated, contractile tail region by a constricted neck region. The head capsules ranged in diameter between 90-110nm with the tail length ranging from 150-200nm in the non-contractile state and 100-130nm in the contractile state. Rigid tail fibres were also visible below the striated tail. The major steps in the virus replicative cycle were also documented as electron micrographs. Ultra-thin sections through phage plaques were prepared through a modification of traditional methods to speed up the process, with no negative effects on sample integrity. The major steps that were captured in the phage replicative cycle were (1) attachment to host cells, (2) replication within host cells, and, (3) release from cells. Overall results suggested that all three phages are strains from the order Caudovirales and are part of the Myoviridae family. A wealth of information can be derived about an organism based on analysis of its proteomic data. In the current study, one-dimensional electrophoretic methods, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and ultra-thin layer isoelectric focusing (UTLIEF), were used to analyse the proteins of three phages, Sabp-P1, Sabp-P2 and Sabp-P3, in order to determine whether these strains differed from each other. SDS-PAGE analysis produced unique protein profiles for each phage, with band fragments ranging in size from 8.86-171.66kDa. Combined similarity matrices showed an 84.62% similarity between Sabp-P1 and Sabp-P2 and a 73.33% similarity between Sabp-P1 and Sabp-P3. Sabp-P2 showed a 69.23% similarity to Sabp-P3. UTLIEF analysis showed protein isoelectric charges in the range of pI 4.21-8.13, for all three phages. The isoelectric profiles for each phage were distinct from each other. A combined similarity matrix of both SDS-PAGE and UTLIEF data showed an 80.00% similarity between phages Sabp-P1 and Sabp-P2, and a 68.29% similarity between Sabp-P1 and Sabp-P3. Sabp-P2 showed a 70.59% similarity to Sabp-P3. Although the current results are based on putative protein fragments analysis, it can be confirmed that phages Sabp-P1, Sabp-P2 and Sabp-P3 are three distinct phages. This was further confirmed through genomic characterization of the three staphylococcal phages, Sabp-P1, Sabp-P2 and Sabp-P3, using restriction fragment length analysis and whole genome sequencing. Results showed that the genomes of phages Sabp-P1, Sabp-P2 and Sabp-P3 were all different from each other. Phages Sabp-P1 and Sabp-P3 showed sequence homology to a particular form of Pseudomonas phages, called "giant" phages. Phage Sabp-P3 showed sequence homology to a Clostridium perfringens phage. Major phage functional proteins (the tail tape measure protein, virion structural proteins, head morphogenesis proteins, and capsid proteins) were identified in all three phages. However, although the level of sequence similarity between the screened phages and those already found on the databases, enabled preliminary classification of the phages into the order Caudovirales, family Myoviridae, the level of homology was not sufficient enough to assign each phage to a particular type species. These results suggest that phage Sabp-P1 might be a new species of phage within the Myoviridae family. One longer-term objective of the study is to carry out complete assembly and annotation of all the contigs for each phage. This will provide definitive conclusions in terms of phage relatedness and classification. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.