Summary: Mycoplasma mycoidessubsp.capri(Mmc) and subsp.mycoides(Mmm) are important ruminant pathogens worldwide causing diseases such as pleuropneumonia, mastitis and septicaemia. They express galactofuranose residues on their surface, but their role in pathogenesis has not yet been determined. TheM. mycoidesgenomes contain up to several copies of theglfgene, which encodes an enzyme catalysing the last step in the synthesis of galactofuranose. We generated a deletion of theglfgene in a strain ofMmcusing genome transplantation and tandem repeat endonuclease coupled cleavage (TREC) with yeast as an intermediary host for the genome editing. As expected, the resultingYCp1.1‐Δglfstrain did not produce the galactofuranose‐containing glycans as shown by immunoblots and immuno‐electronmicroscopy employing a galactofuranose specific monoclonal antibody. The mutant lacking galactofuranose exhibited a decreased growth rate and a significantly enhanced adhesion to small ruminant cells. The mutant was also ‘leaking’ as revealed by a β‐galactosidase‐based assay employing a membrane impermeable substrate. These findings indicate that galactofuranose‐containing polysaccharides conceal adhesins and are important for membrane integrity. Unexpectedly, the mutant strain showed increased serum resistance.

Contagious caprine pleuropneumonia (CCPP), caused by Mycoplasma capricolum subsp. capripneumoniae is a severe disease widespread in Africa and Asia. Limited knowledge is available on the pathogenesis of this organism, mainly due to the lack of a robust in vivo challenge model and the means to do site-directed mutagenesis. This work describes the establishment of a novel caprine challenge model for CCPP that resulted in 100% morbidity using a combination of repeated intranasal spray infection followed by a single transtracheal infection employing the recent Kenyan outbreak strain ILRI181. Diseased animals displayed CCPP-related pathology and the bacteria could subsequently be isolated from pleural exudates and lung tissues in concentrations of up to 109 bacteria per mL as well as in the trachea using immunohistochemistry. Reannotation of the genome sequence of ILRI181 and F38T revealed the existence of genes encoding the complete glycerol uptake and metabolic pathways involved in hydrogen peroxide (H2O2) production in the phylogenetically related pathogen M. mycoides subsp. mycoides. Furthermore, the expression of L-α-glycerophosphate oxidase (GlpO) in vivo was confirmed. In addition, the function of the glycerol metabolism was verified by measurement of production of H2O2 in medium containing physiological serum concentrations of glycerol. Peroxide production could be inhibited with serum from convalescent animals. These results will pave the way for a better understanding of host–pathogen interactions during CCPP and subsequent vaccine development.

Capsular polysaccharides have been confirmed to be an important virulence trait in many gram-positive and gram-negative bacteria. Similarly, they are proposed to be virulence traits in minimal Mycoplasma that cause disease in humans and animals. In the current study, goats were infected with the caprine pathogen Mycoplasma mycoides subsp. capri or an engineered mutant lacking the capsular polysaccharide, galactofuranose. Goats infected with the mutant strain showed only transient fever. In contrast, 5 of 8 goats infected with the parental strain reached end-point criteria after infection. These findings confirm that galactofuranose is a virulence factor in M. mycoides.

Members of 'Mycoplasma mycoides cluster' are important ruminant pathogens in Africa. Diseases caused by these Mycoplasma negatively affect the agricultural sector especially in developing countries through losses in livestock productivity, mortality and international trade restrictions. There is therefore urgent need to develop antimicrobials from alternative sources such as medicinal plants to curb these diseases. In Kenya, smallholder farmers belonging to the Maasai, Kuria and Luo rely on traditional Kenyan herbals to treat respiratory symptoms in ruminants. In the current study extracts from some of these plants were tested against the growth of members of Mycoplasma mycoides cluster. This study aimed at identifying plants that exhibit antimycoplasmal activities using an ethnobotanical approach. Kenyan farmers of Maasai, Luo and Kuria ethnic groups were interviewed for plant remedies given to livestock with respiratory syndromes. The plant materials were thereafter collected and crude extracts prepared using a mixture of 50% of methanol (MeOH) in dichloromethane (CH2Cl2), neat methanol (MeOH), ethanol (EtOH) and water to yield four crude extracts per plant part. The extracts were tested in vitro against five strains of Mycoplasma mycoides subsp. capri, five strains of Mycoplasma mycoides subsp. mycoides and one strain of Mycoplasma capricolum subsp capricolum using broth micro-dilution assays with an initial concentration of 1mg/ml. Minimum inhibitory concentration (MIC) of the most active extracts were determined by serial dilution. Extracts from five plants namely: Solanum aculeastrum, Albizia coriaria, Ekebergia capensis, Piliostigma thonningii and Euclea divinorum exhibited the highest activities against the Mycoplasma strains tested. Mycoplasma mycoides subsp. mycoides were more susceptible to these extracts than Mycoplasma mycoides subsp. capri and Mycoplasma capricolum susp. capricolum. The activities of the crude extracts varied with the solvent used for extraction. The MICs mean values of the active extracts varied from 0.02 to 0.6mg/ml. The results suggested that these plants could potentially contain antimicrobial compounds that might be useful for the treatment of respiratory diseases in ruminants. Future work should focus on the isolation and identification of the active compounds from the plant extracts that showed interesting activities and evaluation of their antimicrobial and cytotoxic potential.