Hezron Wesonga

Assessment of in vitro interferon-γ responses from peripheral blood mononuclear cells of cattle infected with Mycoplasma mycoides ssp. mycoides small colony type

Contagious bovine pleuropneumonia (CBPP) is a lung disease caused by the bacterial pathogen Mycoplasma mycoides ssp. mycoides small colony type (MmmSC). It has been spreading due to a number of factors including poor vaccine efficacy and poor sensitivity of current diagnostic tests. The purpose of this study was to assess interferon gamma (IFN-gamma) release after stimulation of peripheral blood mononuclear cells (PBMC) from experimentally infected cattle. PBMC collected from 15 artificially infected animals were incubated with different concentrations of total MmmSC antigen. After 72h of incubation the IFN-gamma release was measured and found to be elevated in 11 animals. We did not observe a correlation between IFN-gamma release of animals with and without pathomorphological gross lesions. Therefore, our data do not confirm a role for CD4 T-lymphocytes in protection, since there is no correlation between IFN-g secretion (supposed to be mainly derived from CD4 T-cells) and disease severity. Additionally, we applied immunocytochemistry on affected lung tissue and detected no build up of T-lymphocytes (CD4 T-cells, CD8 T-cells) but a high presence of myeloid cells.

Efficacy of two vaccine formulations against contagious bovine pleuropneumonia (CBPP) in Kenyan indigenous cattle

A live, attenuated vaccine is currently the only viable option to control of CBPP in Africa. It has been suggested that simple modifications to current vaccines and protocols might improve efficacy in the field. In this report we compared the current vaccine formulation with a buffered preparation that maintains Mycoplasma viability at ambient temperature for a longer time. Groups of animals were vaccinated with the two formulations and compared with non vaccinated groups. Half of the animals in each group were challenged 3 months post vaccination, the other half after 16 months. Protection levels were measured using the pathology index, calculated from post mortem scores of lesions from animals killed during the course of clinical disease. In the challenge at 3 months post vaccination, the protection levels were 52% and 77% for the modified and current vaccine preparations, respectively. At 16 months post vaccination, the protection levels were 56% and 62% for the modified and current vaccine preparations, respectively. These findings indicate that there are no differences in protection levels between the two vaccines. Because of its longer half life after reconstitution, the modified vaccine might be preferred in field situations where the reconstituted vaccine is likely not to be administered immediately.

Vaccination of Cattle with the N Terminus of LppQ of Mycoplasma mycoides subsp. mycoides Results in Type III Immune Complex Disease upon Experimental Infection

ABSTRACT Contagious bovine pleuropneumonia (CBPP) is a serious respiratory disease of cattle caused by Mycoplasma mycoides subsp. mycoides. Current vaccines against CBPP induce short-lived immunity and can cause severe postvaccine reactions. Previous studies have identified the N terminus of the transmembrane lipoprotein Q (LppQ-N′) of M. mycoides subsp. mycoides as the major antigen and a possible virulence factor. We therefore immunized cattle with purified recombinant LppQ-N′ formulated in Freunds adjuvant and challenged them with M. mycoides subsp. mycoides. Vaccinated animals showed a strong seroconversion to LppQ, but they exhibited significantly enhanced postchallenge glomerulonephritis compared to the placebo group (P = 0.021). Glomerulonephritis was characterized by features that suggested the development of antigen-antibody immune complexes. Clinical signs and gross pathological scores did not significantly differ between vaccinated and placebo groups. These findings reveal for the first time the pathogenesis of enhanced disease as a result of antibodies against LppQ during challenge and also argue against inclusion of LppQ-N′ in a future subunit vaccine for CBPP.

Cattle immunized against the pathogenic L-α-glycerol-3-phosphate oxidase of Mycoplasma mycoides subs. mycoides fail to generate neutralizing antibodies and succumb to disease on challenge.

Highlights • Cattle were vaccinated with Mycoplasma mycoides mycoides (Mmm) recombinant l-α-glycerol-3-phosphate oxidase.• A mouse mAb to l-α-glycerol-3-phosphate oxidase was generated.• Mouse mAb blocked H2O2 release by Mmm, but cattle antisera did not.• Cattle were not protected after challenge.

High quality draft genomes of the Mycoplasma mycoides subsp. mycoides challenge strains Afadé and B237.

Members of the Mycoplasma mycoides cluster’ represent important livestock pathogens worldwide. Mycoplasma mycoides subsp. mycoides is the etiologic agent of contagious bovine pleuropneumonia (CBPP), which is still endemic in many parts of Africa. We report the genome sequences and annotation of two frequently used challenge strains of Mycoplasma mycoides subsp. mycoides, Afadé and B237. The information provided will enable downstream ‘omics’ applications such as proteomics, transcriptomics and reverse vaccinology approaches. Despite the absence of Mycoplasma pneumoniae like cyto-adhesion encoding genes, the two strains showed the presence of protrusions. This phenotype is likely encoded by another set of genes.

Assessing the effectiveness of intubation as a challenge model in contagious bovine pleuropneumonia vaccine experiments

A study was carried out to assess the effectiveness of a bronchoscope in administering a pathogenic field strain of Mycoplasma mycoides subsp. mycoides (MmmSC) in cattle challenge experiments. Out of 16 animals inoculated using the bronchoscope, 10 (62.2%) showed clinical disease as evidenced by fever and 15 (93.8%) displayed typical lesions of CBPP from which MmmSC was isolated. Serum samples collected weekly were tested by Complement Fixation Test (CFT) and competitive enzyme-linked immunosorbent assay (c-ELISA). Antibodies to MmmSC were detected in 10 out of the 16 animals by the CFT and 11 out of the 16 animals by c-ELISA. The onset of clinical disease was as early as 2xa0days post-inoculation, and most of the animals developed clinical disease 2 to 3xa0weeks post-infection. These results clearly demonstrate that nasotracheal inoculation of pathogenic strain of MmmSC with the aid of a bronchoscope can lead to early onset of clinical disease; similar to previous studies but with higher numbers of animals showing clinical disease. This is in contrast with previous studies where early clinical disease was observed in as little as 15% of inoculated animals. This nasotracheal inoculation method using a bronchoscope can, therefore, be adopted for use in experimental challenge infections of cattle. This method is found to be a better replacement to the contact transmission method whose drawback includes extra cost of donor animals and unpredictable rate and timing of transmission from intubated to challenge animals.

Experimental evaluation of inactivated and live attenuated vaccines against Mycoplasma mycoides subsp. mycoides

The current control method for contagious bovine pleuropneumonia (CBPP) in Africa is vaccination with a live, attenuated strain of Mycoplasma mycoides subsp. mycoides (Mmm). However, this method is not very efficient and often causes serious adverse reactions. Several studies have attempted to induce protection using inactivated mycoplasma, but with widely contradictory results. Therefore, we compared the protective capacity of the live T1/44 vaccine with two inactivated preparations of Mmm strain Afadé, inoculated with an adjuvant. Protection was measured after a challenge with Afadé. The protection levels were 31%, 80.8% and 74.1% for the formalin-inactivated, heat-inactivated and live attenuated preparations, respectively. These findings indicate that low doses of heat-inactivated Mmm can offer protection to a level similar to the current live attenuated (T1/44) vaccine formulation.

Capsular polysaccharide from Mycoplasma mycoides subsp. mycoides shows potential for protection against contagious bovine pleuropneumonia

Contagious Bovine Pleuropneumonia (CBPP) is a severe respiratory disease caused by Mycoplasma mycoides subsp. mycoides (Mmm) which is widespread in Africa. The capsule polysaccharide (CPS) of Mmm is one of the few identified virulence determinants. In a previous study, immunization of mice against CPS generated antibodies, but they were not able to prevent multiplication of Mmm in this model animal. However, mice cannot be considered as a suitable animal model, as Mmm does not induce pathology in this species. Our aim was to induce antibody responses to CPS in cattle, and challenge them when they had specific CPS antibody titres similar or higher than those from cattle vaccinated with the live vaccine. The CPS was linked to the carrier protein ovalbumin via a carbodiimide-mediated condensation with 1-ethyl-3(3-imethylaminopropyl) carbodiimide (EDC). Ten animals were immunized twice and challenged three weeks after the booster inoculation, and compared to a group of challenged non-immunized cattle. When administered subcutaneously to adult cattle, the vaccine elicited CPS-specific antibody responses with the same or a higher titre than animals vaccinated with the live vaccine. Pathology in the group of immunized animals was significantly reduced (57%) after challenge with Mmm strain Afadé compared to the non-immunized group, a figure in the range of the protection provided by the live vaccine.

Prevalence of contagious caprine pleuro-pneumonia in pastoral flocks of goats in the Rift Valley region of Kenya.

A cross-sectional survey was conducted between the months of March 2014 and March 2015 to determine the prevalence of contagious caprine pleuropneumonia in goat populations in pastoral flocks in three sub-counties of the Rift Valley region. A total of 432 serum samples were collected from goats from 54 flocks and tested for the presence of antibodies against mycoplasma capricolum subspecies capripneumoniae (mccp) using monoclonal antibody-based competitive enzyme-linked immuno-sorbent assay. Sero-prevalence recorded for Turkana West was 63.9xa0%, Kajiado Central was 48.6xa0%, while Pokot East was 29.2xa0% which was statistically significant (χ2u2009=u200934.997; Pu2009=u20090.000) in the study sites. The results of this study confirmed that CCPP is widespread and endemic in the pastoral production systems studied in the Rift Valley region. The results confirmed that regions sharing international boundaries are at a higher risk of CCPP hence the need for a unified cross-border approach to disease control measures in the border areas.

Risk factors associated with contagious caprine pleuro-pneumonia in goats in pastoral areas in the Rift Valley region of Kenya

A cross-sectional study to determine risk factors associated with sero-prevalence of contagious caprine pleuro-pneumonia (CCPP) in goats was carried out between the months of March, 2014 and March, 2015 in Pokot East, Turkana West and Kajiado Central Sub-counties. A semi-structured questionnaire focusing on risk factors for CCPP was completed for each flock whose serum samples were collected. A logistic regression model was developed to assess the association between the risk factors and CCPP sero-positivity. Of the 54 flocks, 49 (90.7%) presented at least one sero-positive animal. Two hundred and four of the 432 goats tested sero-positive at monoclonal antibody based competitive Enzyme-linked immuno-sorbent assay (c-ELISA), hence a sero-prevalence of 47.2% (95% CI=42.5-u200951.9). Previous exposure of flocks to CCPP (p<0.001, OR=52.8; CI=6.45, 432), distant sources of veterinary drugs (p<0.001, OR=6.17; CI=3.41, 11.1), movement of goats to dry season feeding areas (p<0.001, OR=4.31; CI=2.39, 7.75) and markets as a source of new introductions to the flock (p=0.033, OR=1.86; CI=1.05, 3.27) were identified as risk factors significantly associated with CCPP sero-prevalence. The findings provide further evidence supporting the high prevalence and endemic state of the disease in pastoral flocks and hence there is need for adequate measures to be put in place to control the disease effectively.