Thesis (Ph. D.)--University of Wisconsin--Madison, 1955. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 43-46).

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 26-32).

Thesis (M.S.)--University of Wisconsin--Madison, 1965. / eContent provider-neutral record in process. Description based on print version record. Bibliography: l. 58-62.

Thesis (M.Med.Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 26-32). Also available in print.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Infectious disease continues to cause significant problems on reproductive efficiency in the cattle industry. The purpose of this project is to evaluate new testing strategies for Tritrichomonas foetus and Campylobacter fetus subsp. venerealis. This thesis describes the result of three studies that evaluated the use of real-time PCR for the identification of Tritrichomonas foetus and Campylobacter fetus subsp. venerealis in carrier bulls. The first study evaluated the specificity of a real-time PCR test for T. foetus in individual culture enriched samples, and the sensitivity of the assay for use in pooled samples of up to 25 bulls. Specificity estimates were 98.8% (95% CI 97-99.4) and 100% (95% CI 98.9-100) for culture and real-time PCR, respectively. The sensitivity of the real-time PCR assay for pooled preputial samples was: 96.8% (83.8-99.4) for pool ratios 1/3 and 1/5; 93.5% (79.3-98.2) for pool ratios 1/2, 1/15, 1/20 and 1/25; and 90.3% (75.1-96.6), and were not significantly different. However, 13 of the 217 pools tested were negative and 9 of these negative testing pools contained the same positive sample. The media in this positive sample showed evidence of contamination and could potentially explain the failure to detect T. foetus. The second study evaluated the sensitivity of a real-time PCR for the detection of T. foetus in individual and pooled direct preputial samples. Sensitivity of individual samples tested by culture, real-time PCR in direct and culture enriched samples were determined from 121 samples obtained from 9 infected bulls. Sensitivity estimates were: 95.0% (95% CI: 89.6% to 97.7%) for culture, 95.9% (95% CI: 90.7 to 98.2) for real-time PCR in cultured enriched samples, and 90.1% (95% CI: 83.5 to 94.2) for direct preputial samples and did not differ (P=0.12). Sensitivity estimates for direct pooled samples in groups of 5 or 10 were: 83.6% (95% CI: 75.6 to 89.4) and 77.3% (95% CI: 68.6-84.1), respectively and were not significantly different (P=0.08). The use of repeat sampling tested in pools by real-time PCR increased the sensitivity to 100% and 96% for 3 consecutive samples (pools of 5 or 10, respectively). The use of pooled direct preputial samples although sensitive, still requires the use of repeated sampling. The third study determined the sensitivity and specificity of a recently developed real-time PCR (qPCR) tests for Cfv. A total of 300 virgin bulls were tested by both culture and qPCR. Specificity estimates were 85% (95% CI: 80.5 to 88.6) for qPCR and 100% (95% CI: 98.7 to 100) for culture, and were significantly different (P<0.01). A total of 4 naturally infected bulls and 9 artificially infected bulls were sampled serially to obtain positive samples for a sensitivity analysis. Sensitivity estimates and 95% confidence intervals are as follows: qPCR (85.4%, 95% CI: 80.6-89.2); direct culture on blood agar (82.3%, 95% CI: 77.2-86.5), DFAT (72.1%, 95% CI: 66.2-77.4), direct culture on Skirrow agar (32.7%, 95% CI: 27.2-38.7), TEM and blood agar (30%, 95% CI: 23.4-37.5), and TEM and Skirrow agar (38.1%, 95% CI: 31-45.9). The sensitivity of the different tests evaluated varied significantly with different ambient temperatures (P<0.01). The sensitivity of the qPCR was significantly higher than any other test when temperatures exceeded 5°C. The use of repeated sampling at weekly intervals significantly improved the sensitivity of the qPCR. The real-time PCR assay for the detection of T. foetus in both individual and pooled samples appears to be highly sensitive and specific. Moreover, the possibility of using direct preputial samples provides a cost-effective diagnostic strategy. Real-time PCR in direct preputial samples for BGC diagnosis in bulls has good sensitivity and specificity. However, the use of repeated sampling maybe needed in order to maximize the ability to detect carrier bulls.

Using the chemically defined medium designed by Smibert (1963), the stimulatory or inhibitory effect of test compounds on the growth and the production of acid precipitable material by seven strains of vibrio fetus was determined. The test compounds were Krebs cycle intermediates, lactate, pyruvate, fatty acid, amines, surface active agents, aminobutyrate, hydroxybutyrate, glutathione, asparagine, phenylacetyl chloride, and sodium bicarbonate. Experimental media consisted of the chemically defined medium containing various concentrations of the test compounds. An exact number of cells of each strain of V. fetus was inoculated into tubes of experimental broth media. The cultures were incubated at 37 °C in desiccator jars in an atmosphere containing 85% nitrogen, 10% carbon dioxide, and 5% oxygen. Cultures were observed daily for visible growth. After five days incubation, the optical density of each culture was read at 5/+0 mμ; in a spectrophotometer. The concentration of APM produced by each strain of V. fetus grown in experimental media was also determined. Five day old cultures of each strain were centrifuged, and the APM present in the supernatant fluid was precipitated with trichloroacetic acid. The optical density of the mixture was read at 380 mμ, in the spectrophotometer. Lactate increased the growth of four of five strains of V. fetus tested, and did not inhibit the growth of any strains. Lactate increased APM production of V. fetus due to the increased number of cells produced in its presence. Therefore, the addition of lactate to the 8 chemically defined mediums used for growth or APM production would be advantageous. Citrate and succinate, which increased the growth of V. fetus without affecting APM production, could be added to the chemically defined medium used for obtaining maximal growth of vibrios. Studies requiring a large cell crop of vibrios would be facilitated by the use of the chemically defined medium to which lactate, citrate, and/or succinate had been added. A large cell crop of V. fetus would be useful in preparation of V. fetus cellular antigen. Any study requiring large cell crops cf vibrios would be facilitated by the use of a medium which increased the growth of V. fetus. Alpha-ketoglutarate and tween 80 increased APM production of V. fetus, although these compounds neither increased nor decreased the growth of the vibrios. Therefore, the addition of α-ketoglutarate and tween 80 to the medium used for maximal production of APM would be advantageous. An increased production of APM by V. fetus would aid the study of the biosynthesis of APM. Also, the antigenic nature of APM could be studied more successfully by using a medium which increased the production of APM by vibrios. Compounds, such as glutathione and tergitol 7, which inhibited the growth and the production of APM of vibrios should not be added to the chemically defined medium. Glutamine and asparagine inhibited the growth of V. fetus and should also be avoided as additions to the medium. / Master of Science

Bovine genital campylobacteriosis is a disease of difficult diagnostic because of the microaerophilic nature of its etiologic agent, Campylobacter fetus, thereby causing the prevalence related to this disease to be underestimated. The purpose of this study was to evaluate the utilization of PCR for the diagnosis of genital campylobacteriosis, using samples obtained from bull prepuce aspirate, cow cervical mucus, and abomasum contents obtained from aborted fetuses, collected in transport and enrichment medium. Five different DNA extraction protocols were tested: thermal extraction, lyses with proteinase K, lyses with guanidine isothiocyanate, lyses with DNAzol? , and lyses with CTAB. The specificity, sensitivity and technical application of PCR assay were also evaluated with clinical samples. The PCR performance was compared to the culture technique for bacterial isolation. The CTAB was the most efficient extraction protocol; the pair of primers used was shown to be specific; and the limit of detection was of 63 CFU of Campylobacter fetus. PCR demonstrated that 24% (68/277) of the clinical samples were positive for Campylobacter fetus, while only 2.8% (8/277) of samples were positive by culture techinque. These results indicate that the PCR technique is specific and sensitivy, and is superior to the culture for the diagnosis of bovine genital campylobacteriosis. / A campilobacteriose genital bovina, caracterizada principalmente por infertilidade, é uma doença de difícil diagnóstico, devido à natureza microaerófila do seu agente etiológico, o Campylobacter fetus, tendo assim sua prevalência subestimada. Este trabalho teve por objetivo avaliar a utilização da técnica de PCR para o diagnóstico da campilobacteriose genital bovina, utilizando amostras de aspirado prepucial, muco cervical e conteúdo abomasal de fetos abortados, coletadas em meio de transporte e enriquecimento; bem como comparar seu desempenho com a técnica do isolamento bacteriano. Foram testados cinco diferentes protocolos de extração de DNA de Campylobacter fetus: termo extração, lise com proteinase K, lise com isotiocianato de guanidina, lise com DNAzol? e lise com brometo de cetiltrimetilamônio (CTAB). Também foram avaliadas a especificidade, a sensibilidade e a aplicação da técnica da PCR em amostras clínicas. Os resultados indicaram que o CTAB foi o protocolo de extração mais eficiente; o par de primers utilizado mostrou-se específico; e o limite de detecção foi 63 unidades formadoras de colônias (UFC) de C. fetus. A PCR encontrou 24% (68/277) das amostras clínicas positivas para C. fetus, enquanto a cultura encontrou 2,8% (8/277). A técnica da PCR é específica e sensível, e mostra-se superior a cultura no diagnóstico da campilobacteriose genital bovina.

Bovine Genital Campylobacteriosis is characterized by early pregnancy loss and temporary infertility in cattle. The purpose of this project was to compare diagnostic approaches to detect Campylobacter fetus subsp. venerealis and evaluate the efficacy of vaccination for Bovine Genital Campylobacteriosis. This thesis describes the results of two studies that compared different sample preparation methods for bovine vaginal mucus for real-time PCR and assessed a commercial vaccine in preventing infection and reproductive loss. The first study compared real-time PCR utilizing different bovine vaginal mucus sample preparation techniques to direct culture. The magnetic bead based protocol demonstrated higher sensitivity (48.4%, P=0.02) and lower specificity (78.9%, P=0.01) than the heat lysis protocol which involved an additional dilution step (Sens=29.4%, Spec=88.2%), but did not differ from the heat lysis protocol without sample dilution (Sens=35.0%, P=0.16; Spec=81.1%, P=0.62). The sample preparation method, designed for bovine preputial samples (Chaban et al. 2012. Can J of Vet Res; 76: 166), did not work well for vaginal mucus. All modifications of that method and magnetic bead based extraction technique had low sensitivity compared to culture probably due to the biophysical properties of vaginal mucus, which could cause loss of targeted DNA during processing, or repeated sample freezing and thawing. Release of DNA directly from vaginal mucus by a modified heat lysis protocol with consequent real-time PCR could be a promising rapid screening approach after validating on fresh samples. The second study compared the risk of infection and reproductive failure in heifers, vaccinated with a commercial multivalent vaccine containing C. fetus antigen, to heifers vaccinated with a comparable product without C. fetus, that were exposed to infected bulls. There was no significant difference between groups either in risk of Campylobacter fetus subsp. venerealis isolation (P>0.17) or in the proportion of heifers that cultured positive at least once (P=0.42), as well as in the median number times of cultured positive samples (P=0.24) and the time to first cultured positive (P=0.67). There was no difference by treatment in the weekly proportions of heifers diagnosed pregnant by either ultrasound (P>0.31) or serum concentration of pragnancy specific protein B (P>0.31) during the study, as well as in the time to first pregnancy for heifers ever diagnosed as pregnant (P=0.30) and those that remained pregnant at the end of the study (P=0.70). Similarly, the difference was not detected by treatment in the proportion of animals, ever detected pregnant during the study (P=0.57) and in pregnancy loss rates (P=0.28). However, heifers that aborted were 4 times more likely to be cultured positive than those that did not abort (P=0.01). Heifers that were not pregnant at the end of the study cultured positive 1.5 times more often than pregnant animals in treatment group (P=0.04), while in control group such difference was 4 times (P=0.01). Heifers that were not pregnant at the end of the study did not differ by treatment in the number of times cultured positive (P=0.14). In this study, the mean concentrations of ELISA antibodies to C. fetus after vaccination were more than 2 times higher in treatment group than in control group (P<0.02), but vaccination did not significantly reduce infection or improve pregnancy in heifers when exposed to Cfv-infected bulls. Sample preparation technique is important for successful real-time PCR; release of DNA directly from a CVM sample by a modified heat lysis protocol was easy to perform and could be promising as a rapid screening approach for Bovine Genital Campylobacteriosis after validating on fresh samples. Vaccinating of heifers with a polyvalent commercial vaccine, containing Campylobacter fetus antigen, according to the label, did not significantly reduce infection rate or improve reproductive performance when they were naturally challenged.

Campylobacter fetus subsp. venerealis ist der Erreger der bovinen genitalen Campylobacteriose, einer anzeigepflichtigen Tierseuche. Er hat sein natürliches Reservoir im Präputialsack klinisch gesunder Bullen und ist in der Lage, Aborte zu verursachen (enzooti-scher Abort). Davon abzutrennen sind Infektionen mit C. fetus subsp. fetus, welcher natürlicherweise im Intestinaltrakt des Rindes auftritt und ebenfalls Aborte auszulösen vermag (spo-radischer Abort). Während C. fetus subsp. venerealis einen ausgeprägten Tropismus für die Genitalschleimhaut des Rindes aufweist, handelt es sich bei der Subspezies fetus um einen Zoonoseerreger, der beim Menschen schwerwiegende Erkrankungen mit zumeist septikämischen Charakter verursachen kann. Die Übertragung von C. fetus subsp. venerealis erfolgt hauptsächlich durch den natürlichen Deckakt, es besteht aber auch die Gefahr der Verbreitung durch künstliche Besamung, da klinisch gesunde Bullen den Erreger im Samen enthalten können. Die Unterschiede in der Epidemiologie und die klinische Bedeutung der beiden C. fetus-Subspezies erfordern eine exakte Identifizierung und Differenzierung. Der erste Teil der Untersuchung befasst sich daher mit den Möglichkeiten des molekularen Erregernachweises. Im zweiten Teil wurden 50 Isolate aus den vergangenen fünf Jahren mit molekularen Methoden auf ihre genetische Ähnlichkeit untersucht. Eine Abgrenzung durch traditionelle mikrobiologische Methoden ist sehr problematisch, da sie im Wesentlichen auf nur zwei Reaktionen beruht: die Glycin-Toleranz und die Na-Selenit-Reduktion, wobei neueste Untersuchungen unter standardisierten Bedingungen nur die Glycin-Toleranz als eindeutig charakterische Reaktion für beide Subspezies übrigließe. Aus diesem Grunde wurden PCR-Untersuchungen zur Identifizierung und Differenzierung beider C. fetus-Subspezies eingeführt. In einem ersten Schritt wurde Campylobacter fetus spezifisch nachgewiesen. Danach erfolgte die Differenzierung der Subspezie durch eine weitere PCR, in der nur bei Vorliegen der DNS von C. fetus subsp. venerealis ein Amplikon erhalten wurde. Insgesamt wurden 103 C. fetus-Isolate untersucht, einschließlich der Typenstämme von C. fetus subsp. fetus und C. fetus subsp. venerealis. Auf Grund der Ergebnisse des Gly-cintoleranztests konnten 81 C. fetus subsp. venerealis (Glycin intolerant) und 22 C. fetus subsp. fetus (Glycin tolerant) identifiziert werden. Die Ergebnisse des Glycintoleranztests und der PCR stimmten bei allen 103 C. fetus Isolaten überein. Versuche zum Direktnachweis von C. fetus subsp. venerealis aus Bullensperma durch Anwendung von fünf unterschiedlichen DNS-Extraktionsmethoden waren in ihren Ergebnissen hinsichtlich ihrer Sensitivität nicht zufrieden stellend (104 KbE/ml). Daraus ergibt sich zwingend, dass die Kultivierung des Erregers vor seiner phäno- und genotypischen Charakterisierung weiterhin unverzichtbar bleibt. Durch Untersuchungen mittels PFGE wurde gezeigt, dass die Campylobacter fetus subsp. venerealis-Population, die in Deutschland vorkommt, genetisch nicht einheitlich ist. Eine strenge Gruppierung nach geografischen Regionen war nicht möglich. Innerhalb größerer Gruppen genetisch ähnlicher Stämme fielen Isolate mit identischen Mustern auf, was auf gemeinsame Infektionsquellen hindeutet. Die ERIC-PCR sollte in der vorliegenden Arbeit als zweite Methode der Analyse des Gesamtgenoms zur weiteren Stützung der Ergebnisse der Makrorestriktionsanalyse beitragen. Die Analysen mittels ERIC-PCR machte ein hohes Maß an Heterogenität innerhalb der einzelnen Spezies sichtbar, lässt jedoch keine Assoziation zwischen Bandenprofilen und einer Zuordnung zum Krankheitsbild oder zur geographischen Herkunft zu.