Mycoplasmas are responsible for many important diseases of animals, including contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides SC and calf pneumonia, arthritis and mastitis caused by Mycoplasma bovis. However, diagnostic techniques currently available are laborious and imprecise. The work described in this thesis concentrates on the critical evaluation of existing techniques and the development of improved procedures. The role of antimicrobial resistance in limiting the options for disease control was also considered. Diagnostic methods for the detection of M. mycoides subsp. mycoides SC in clinical material were critically evaluated during a CBPP outbreak in Portugal. Immunoblotting was more sensitive and specific than CFT or ELISA. The polymerase chain reaction (PCR) was more rapid and sensitive than culture. However immunocytohistochernistry (ICC) was far the best test for detecting M. mycoides subsp. mycoides SC antigen in lungs. A rapid latex agglutination test (LAT) to detect CBPP using a carbohydrate extract of M. mycoides subsp. mycoides SC was developed. Analysis of the carbohydrate extract composition demonstrated that fucose, glucosamine and galactose are present in the ratio of 1:2:16 respectively. N-acetyl neuraminic acid was also detected. Evaluation of the LAT with sera from negative, naturally infected and experimentally infected cattle demonstrates that the test clearly differentiates positive and negative CBPP sera. The LAT compared favourably with the CFT but was not as specific as the immunoblotting; however the LAT had the advantage of being more rapid and robust and could be used in the field. Molecular methods including the polymerase chain reaction (PCR) and 16S rRNA gene sequencing were assessed for their potential use in the diagnostic laboratory. A PCR method for identifying M. bovis was adapted, evaluated and introduced as a routine laboratory test. Using a set of universal 16S rRNA gene primers, amplicons of two serologically untypable isolates, one from a peregrine falcon, and the other from an ostrich were obtained. Results imply that the isolates may be new mycoplasma species. The development of antimicrobial resistance has been seen in many microorganisms but little evidence exists for resistance in mycoplasmas. Consequently, the in vitro effect of five antimicrobials; danofloxacin, oxytetracycline, spectinomycin, florfenicol and tilmicosin on 62 isolates of M. bovis and 20 of M. mycoides subsp. mycoides SC was investigated. Nfinimum inhibitory concentrations (MICs) and mycoplasmacidal (MMC) values were determined. Evidence of antimicrobial resistance by M. bovis is shown. The potential for M. mycoides subsp. mycoides SC to develop antimicrobial resistance against spectinomycin in vitro is also demonstrated.

Contagious bovine pleuropneumonia (CBPP) is a bovine bacterial disease of major economic importance in sub-Saharan Africa. Vaccination has been recommended to control the disease in endemic areas such as the Maasai ecosystems of Kenya and Tanzania; however, the currently used live attenuated vaccine has been reported to have poor vaccine safety and efficacy. To compare standard (current) and an improved (buffered) version of the live CBPP-vaccine, several epidemiological studies were carried out in Maasai cattle in Kenya between 2006 and 2008. Specifically, the aims were to estimate CBPP seroprevalence at herd and animal level; to identify risk factors for seroprevalence at both levels; to investigate the spatial distribution of seroprevalence; to compare post vaccination adverse events in cattle vaccinated with a standard and a buffered vaccine, and finally to compare efficacy of the two vaccines to induce seroconversion and to prevent development of clinical signs suggestive of CBPP. A cross-sectional study was carried out in 6872 cattle in 175 randomly selected herds from Loita and Mara divisions. A competitive ELISA revealed that 85% of the herds in the area had at least one seropositive animal and that seropositive herds were harbouring 11% seropositive cattle. A complement fixation test revealed that 46% of the herds had at least one seropositive animal and that seropositive herds were harbouring 4% seropositive cattle. A multivariable logistic regression analysis of the seroprevalence indicated that previous vaccination against CBPP, a history of CBPP outbreaks in the herd, animal age and the location of the herd in the division of Mara were positively correlated to seroprevalence. To investigate the observed difference in herd seroprevalence between the two divisions further, a spatial analysis was conducted. A SatScan test revealed clusters in Mara in areas identified by veterinary personnel as CBPP ‘hot spots’. A logistic regression using spatial information identified that location in the midland agro-ecological zone or close to a river and vaccination were positively associated with seroprevalence. To compare safety and efficacy of a standard and a buffered vaccine, two cohorts of approximately 40,000 cattle were used. The study showed that within 100 days post vaccination, 6.2 cattle per 1000 vaccinates developed adverse events, 4.1 of which were specifically attributable to vaccination and ranging from swelling of the tail to the tail sloughing off. This study revealed a slightly higher incidence of adverse events in cattle vaccinated with the buffered vaccine compared to the standard vaccine. A comparison of the efficacy of the two vaccines revealed that cattle vaccinated with the buffered vaccine had higher odds of seroconversion and lower odds of developing symptoms of CBPP, three and twelve months post vaccination respectively. The epidemiological studies conducted clearly show wide spread seroprevalence in the Maasai cattle. Given the (spatial) heterogeneity observed, control measures should probably be targeted in areas of increased risk (clusters). However, positive association of vaccination and seropositivity call for better diagnostics tests that can differentiate vaccinated from infected animals. Vaccination with buffered vaccine resulted in increased seroconversion, decreased clinical signs indicative of CBPP post vaccination and low seroprevalence post ‘outbreak’. Nevertheless, the increase in adverse events related to the buffered vaccine calls for further research into safer CBPP vaccines.

No description available.

No description available.

Thesis (MMedVet (Production Animal Studies)--University of Pretoria, 2007. / Includes bibliographical references.

Contagious Bovine Pleuropneumonia (CBPP), caused by Mycoplasma mycoides mycoides small colony (MmmSC), is a devastating respiratory disease of cattle in Africa, Asia and the Middle East. Little investigation has been done on molecular disease pathogenesis and host response beyond soluble cytokine detection. This study developed and characterized models for three strains of MmmSC of varying severity. Strains used were Gladysdale, Ondangwa and Shawawa. Samples of bronchoalveolar lavage fluid, bronchial biopsy, nasal epithelial cells and blood were obtained prior to and at weekly time points post-infection. Microarray analysis of RNA extracted from samples revealed host cellular pathways and genes important in the pathogenesis of CBPP, including multiple immune system and inflammatory response pathways. A number of pathways whose influence on disease pathogenesis was not immediately clear were also activated, including pathways involved in amino acid synthesis, fat metabolism, and endocrine hormone responses. Microarray results were confirmed with real-time polymerase chain reaction (RT-PCR) of selected genes. Comparative RT-PCR analysis of selected genes between the three strains of MmmSC revealed genes possibly responsible for differential strain virulence, including interleukins 1B, 6, 8, and 18 and the gene nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (NFKBIA). A similar analysis of selected genes between survivors and nonsurvivors of the virulent Gladysdale strain of MmmSC suggested genes involved in survival, including interleukin 8, calmodulin 2 (CALM2), and NFKBIA. Avenues of additional study were identified.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2002. / Härtill 4 uppsatser.

Advances in molecular methods for analyzing DNA, RNA and proteins in humans as well as in other animals, plants, fungi, bacteria or viruses have greatly increased the resolution with which we can study life’s complexity and dynamics on earth. While genomic, transcriptomic and proteomic laboratory tools for molecular diagnosis of disease are rapidly becoming more comprehensive, the access to such advanced yet often expensive and centralized procedures is limited. There is a great need for rapid and comprehensive diagnostic methods in low-resource settings or contexts where a person can not or will not go to a hospital or medical laboratory, yet where a clinical analysis is urgent. In this thesis, results from development and characterization of novel technologies for DNA and protein microarray analysis are presented. Emphasis is on methods that could provide rapid, cost-effective and portable analysis with convenient readout and retained diagnostic accuracy. The first study presents a magnetic bead-based approach for DNA microarray analysis for a rapid visual detection of single nucleotide polymorphisms. In the second work, magnetic beads were used as detection reagents for rapid differential detection of presence of pestiviral family members using a DNA oligonucleotide microarray with read-out by means of a tabletop scanner or a digital camera. In paper three, autoimmune responses from human sera were detected on a protein autoantigen microarray, again by means of magnetic bead analysis. Here, special emphasis was made in comprehensively comparing the performance of the magnetic bead detection to common fluorescence-based detection. In the fourth study, an immunochromatographic lateral flow protein microarray assay is presented for application in the classification of contagious pleuropneumonia from bovine serum samples. The analysis could be performed within 10 minutes using a table top scanner, and the performance of the assay was shown to be comparable to that of a cocktail ELISA. In the fifth paper, the lateral flow microarray framework is investigated in further detail by means of experiments and numerical simulation. It was found that downstream effects play an important role, and the results further suggest that the downstream binding profiles may find use in simple affinity evaluation. / QC 20100713