Les retards au moment des premières vaccinations est un facteur prédictif d’un statut vaccinal incomplet pendant l’enfance. L’étude Promovac, réalisée en Estrie entre 2010 et 2011, a démontré l’efficacité d’une intervention éducative de promotion de la vaccination délivrée, selon les techniques de l’entrevue motivationnelle (EM), aux mères durant leur séjour post-partum à la maternité du Centre Hospitalier Universitaire de Sherbrooke (CHUS). L’intervention a significativement augmenté leur intention de vaccination (+15%) ainsi que les taux de couvertures vaccinales (CV) à 3, 5 et 7 mois de vie (+3,3%, 5% et 7,4%) de leurs nourrissons. L’objectif du présent projet est d’évaluer l’impact à long terme de l’intervention testée dans le cadre de l’étude Promovac. D’une part, sur les connaissances, les croyances, les attitudes et l’intention de vaccination des mères et d’autre part, sur les taux de CV à 13, 19 et 24 mois de vie de leurs nourrissons ainsi que sur l’ensemble du calendrier vaccinal de la petite enfance. Il s’agit d’une étude pré-expérimentale. L’impact à long terme de l’intervention sur les connaissances, les croyances, les attitudes et l’intention de vaccination des mères a été mesuré à l’aide d’un questionnaire auto-administré adressé par voie postale 48 mois après le séjour en maternité. Les données de vaccination de leurs enfants ont été extraites du registre de vaccination de la Direction de Santé Publique de l’Estrie. Les mères de 1140 enfants ont eu l’intervention (groupe expérimental) et 1249 enfants sont dans le groupe témoin. Un impact persistant de l’intervention a été mis en évidence sur les connaissances, les croyances et les attitudes des mères vis-à-vis de la vaccination. Les taux de CV sont significativement plus élevés à 13, 19 et 24 mois (+6,7%, 10,6% et 5,1%) chez les enfants du groupe expérimental comparativement à celles des enfants du groupe contrôle. L’analyse multivariée de l’ensemble des données de CV, a permis de conclure que les enfants du groupe expérimental ont 5% plus de chance d’avoir un statut vaccinal complet entre 3 à 24 mois comparativement aux enfants du groupe contrôle (79,4 vs 74,3%; RR (IC à 95%) = 1,05 (1,02-1,08), p<0,001). L’étude Promovac est à ce jour la seule étude, basée sur une méthode éducative seule, qui a démontré l’efficacité d’une intervention précoce en maternité sur les premières vaccinations mais aussi sur les vaccinations de la petite enfance. Ce résultat pourrait avoir de grandes implications dans les stratégies de promotion globale de la vaccination.

Master of Science / Department of Clinical Science / Elizabeth Davis / Background: While there is a commercially-available vaccine for Streptococcus equi subsp. equi licensed for the intranasal route of administration, some equine practitioners are administering this vaccine orally despite a lack of evidence for its efficacy by this route of administration. Objectives: To compare systemic and local immune responses following intranasal or oral administration of the USDA-approved, live-attenuated Streptococcus equi subspecies equi vaccine (Pinnacle IN®, Zoetis, Florham Park, New Jersey). Study Design: Experimental, randomized clinical trial Methods: Eight healthy horses with low Streptococcus equi M protein (SeM) titers (<1:1600) were randomly assigned to an intranasal or oral two-vaccine series. SeM-specific serum immunoglobulins G (IgG) and A (IgA) and nasal secretion IgA were assessed using a commercially-available ELISA (Equine Diagnostic Solutions, LLC, Lexington, Kentucky) and a novel magnetic microsphere assay utilizing fluorescence. A general linear mixed models approach was used for statistical data analysis. Results: As expected, intranasal vaccinates showed substantial increases in both serum SeM-specific IgG and IgA levels post-vaccination (P=0.0006 and P=0.007, respectively). Oral vaccinates showed an increase in serum SeM-specific IgG post-vaccination (P=0.0150), though only one-third the magnitude of intranasal vaccinates. Oral vaccinates showed no evidence of change in SeM-specific IgA post-vaccination (P=0.15). Main Limitations: Changes in mucosal antibody responses were not identified in this study which may be related to small change in antibody response, timing of sample collection, or method of nasal secretion collection. Conclusions: Results indicate that intranasal or oral vaccine administration resulted in increased serum SeM-specific IgG, though the magnitude of response differed between routes.

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.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

Cancer is a worldwide health problem and despite advances in traditional treatments i.e. surgery, chemotherapy and radiotherapy, the cure rate remains disappointing for some cancers. Different novel therapeutic strategies are being developed. In this thesis two nontraditional cancer therapy approaches are studied: gene therapy using viral vectors and antitumour vaccination with dendritic cell - tumour cell (DC/TC) hybrids. We have developed a novel ELISPOT titration method for viral vectors that is based on the actual expression of the transgene in target cells. This method was developed with recombinant parvovirus MVM-IL2, but it should be adaptable for other vectors carrying expression cassettes for secreted transgene products for which antibodies are available. The ELISPOT titration method allows for faster and better quantification of transducing units present in vector stocks as opposed to titration by in situ hybridisation (annexe I). The MVMIL2 vector has shown an anti-tumour effect against melanoma in an immunocompetent mouse model (annexe IV). Previous work concerns photodynamic inactivation of adenoviral vectors for biosafety and an in vivo study in which a synergistic effect of antiangiogenesis gene therapy combined with radiotherapy could be shown (annexes V and VI). DC/TC hybrids have been proposed as cancer vaccines for their simultaneous expression of antigen presentation machinery and tumour associated antigens. Hybrids are classically generated by polyethylene glycol (PEG) or electrofusion. These methods however require special skills and equipment and cause rather high cell lethality. Fusion via the expression of viral fusogenic membrane glycoproteins (FMG), such as the vesicular stomatitis virus-G (VSV-G) (annexe III) or the Gibbon ape Leukemia Virus (GaLV) FMG, have recently been described. We have mainly focussed on the latter. Transduction of cells with GaLV-FMG proved to be a limiting step for an efficient generation of hybrids. On the other hand, constitutive expression of GaLV-FMG leads to lethal syncytia formation in human cells. Therefore we developed a novel fusion strategy for the generation of DC/TC cell hybrids that involves the use of a non-human fusogenic cell line that constitutively expresses the GaLV-FMG. With this method we were able to generate reproducible yields of DC/TC triparental hybrids. The formation of tri-parental hybrids via the fusogenic cell line is an interesting alternative to existing DC/TC fusion methods because of its simplicity and its flexibility in the choice of fusion partners, i.e. autologous or allogeneic DCs and tumour cells. Moreover, the tri-parent hybrid system offers the possibility to further enhance the immune response by the addition of transgenes that code for immuno-modulating factors to the fusogenic cell line (annexe II).

Trypanosoma congolense is a protozoan parasite transmitted by tsetse flies. It causes bovine trypanosomosis, the major disease for livestock in sub-Saharan Africa. Control methods include trypanocidal drugs and vector control, but none is fully satisfactory, due to resistance and environmental issues. A method that would have the greatest impact on controlling the disease is vaccination. However, development of a conventional vaccine has been hampered by the mechanism of antigenic variation, which allows the parasite to evade the host’s immune system. An alternative strategy in vaccine design is to target the bioactive compounds released by dead and dying trypanosomes. This approach is termed ‘‘anti-disease’’, and does not affect the survival of the parasite but targets the pathogenic factors released by the trypanosomes. The development of a successful anti-disease vaccine necessitates knowledge of all pathogenic factors involved in the disease process. Several macromolecules, primarily peptidases, have been implicated in the pathogenesis of trypanosomosis. Pyroglutamyl peptidase type I (PGP) was shown to be involved in abnormal degradation of thyrotropin- and gonadotropin-releasing hormones in rodents infected with T. brucei, but to date no data are available on the T. congolense PGP. Molecular cloning and expression in E. coli of the coding sequence of T. congolense PGP, as well as the enzymatic characterisation of the recombinant protein, are reported here, completed by the development of reverse genetics tools for studies of gene function. A 678 bp PCR fragment covering the complete open reading frame of PGP was cloned and sequenced. The deduced amino acid sequence showed 52% and 29% identity with the T. brucei and Leishmania major enzymes respectively. The catalytic residues Glu, Cys and His described in Bacilus amyloliquefaciens PGP are conserved in the T. congolense sequence. PGP was expressed in bacterial systems as a soluble active, 26 kDa enzyme. The recombinant enzyme showed activity specific for the fluorescent substrate pGlu-AMC, with a kcat/Km of 1.11 s-1μM. PGP showed activity in the pH 6.5-10 range, with maximal activity at pH 9.0. The enzyme was strongly inhibited by sulfhydryl-blocking reagents such as iodoacetic acid and iodoacetamide with a kass of 125 M-1 s-1 and 177 M-1 s-1 respectively. Antibodies raised in chickens against the recombinant enzyme allowed the detection of native PGP in both procyclic and bloodstream T. congolense developmental stages, and displayed complete inhibition of the enzyme in vitro at physiological concentrations. To get insight into the role of PGP in parasite biology and trypanosomosis progression, two types of vectors for reverse genetics studies were developed. For RNA interference, a 400 bp 3′ end segment of the PGP open reading frame was cloned into the plasmid p2T7Ti, that will allow PGP gene down-regulation upon integration into the genome of an engineered tetracycline-inducible strain such as TRUM:29-13. For gene knock-out, several rounds of molecular engineering were carried-out in order to create two plasmid vectors, pGL1184-based (blasticidin resistance) and pGL1217-based (neomycin resistance), each bearing 200 bp-long regions at the 5′ and 3′ ends of the PGP open reading frame. In subsequent studies, taking advantage of the recent advances in culture and transformation of T. congolense, these plasmids will allow the creation of single and double knock-out mutants of PGP. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Raktažodžiai: gydytojų požiūris, nuomonė, vakcinacija Pagrindimas: Lietuvoje atlikta labai mažai tyrimų apie medikų požiūrį į vakcinaciją, tačiau daugėjant žiniasklaidoje medicininį išsilavinimą turinčių specialistų skeptiškų pasisakymų apie vakcinaciją, tampa aišku, kad ši sritis paveikta tam tikrų mitų apie vakcinaciją ir antivakcininių judėjimų mados tampa problematiška. Tyrimo tikslas: nustatyti Vilniaus poliklinikose dirbančių gydytojų požiūrį į vakcinaciją. Tyrimo uždaviniai: 1. Įvertinti gydytojų požiūrį į vakcinacijos pagrįstumą, infekcinės ligos rizikos suvokimą, vakcinacijos saugumą, efektyvumą, būtinumą, naudingumą, vaikų skiepijimą pagal skiepų kalendorių 2. Įvertinti gydytojų požiūrį į vakcinaciją: nuo difterijos, stabligės, pneumokokinės infekcijos, gripo bei žmogaus papilomos viruso 3. Įvertinti veiksnius galinčius turėti įtakos medikų požiūriui į vakcinaciją. Metodai: iš Vilniaus miesto poliklinikų atsitiktinės atrankos būdu atrinktos 7 poliklinikos, kuriose anketiniu būdu apklausti įvairių specializacijų gydytojai. Duomenų analizei panaudotos 393 užpildytos anketos. Duomenų analizė atlikta SPSS 16.0 for Windows, naudojant aprašomąją statistiką, neparametrinius ir parametrinius kriterijus, bei binarinę logistinę regresiją. Rezultatai: kad vakcinacija nepakankamai pagrįsta moksliniais tyrimais sutiko 86 (24,00%) apklaustieji. Kad vakcinacija sukelia daugiau sveikatos problemų nei nuo jų apsaugo sutiko tik 29 (7,60%) apklaustieji. Kad persirgti infekcine liga... [toliau žr. visą tekstą] / Problem of the study: There is very little research on the medical approach to vaccination in Lithuania, but the increasing number of statements of specialists with medical education with skeptical remarks about the vaccination in media, it becomes clear that their approach to vaccination is affected by a number of myths about vaccination and that this area is problematic. Aim of the study: identification of the attitude to vaccination of doctors working in the outpatient clinics of Vilnius. Tasks of the study: 1. Evaluate the attitude of the physitians to safety, benefit, efficiency of vaccination, and the calendar of children’s vaccination. 2. Evaluate the attitude of the physitians to vaccination against: diphtheria, tetanus, pneumococcal infection, flu, human papillomavirus infection. 3. Evaluate the factors capable to have influence on the attitude of the physitians to vaccination. Methods: 7 outpatient clinics were randomly selected out of outpatient clinics located in city of Vilnius. A special questionnaire was composed for this purpose and doctors of various fields of specializations were questioned. 393 questioners were processed for analysis of the data. Data analysis was performed using 16.0 for Windows, implementing descriptive statistics, nonparametric and parametric criteria, as well as binary logistic regression. Results: 86 (24.00%) agreed that vaccination is not sufficiently evidence based. Only 29 (7.60%) agreed that vaccination induces more related health... [to full text]