Galactofuranose in Mycoplasma mycoides is important for membrane integrity and conceals adhesins but does not contribute to serum resistance.

Mycoplasma mycoides subsp. 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. The M. mycoides genomes contain up to several copies of the glf gene, which encodes an enzyme catalysing the last step in the synthesis of galactofuranose. We generated a deletion of the glf gene in a strain of Mmc using genome transplantation and tandem repeat endonuclease coupled cleavage (TREC) with yeast as an intermediary host for the genome editing. As expected, the resulting YCp1.1-Δglf strain 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 ß-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.

Development of a Novel Cocktail Enzyme-Linked Immunosorbent Assay and a Field-Applicable Lateral-Flow Rapid Test for Diagnosis of Contagious Bovine Pleuropneumonia.

Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease that is widespread in sub-Saharan Africa. It is caused by Mycoplasma mycoides subsp. mycoides, a bacterium belonging to the Mycoplasma mycoides cluster. In the absence of an efficient CBPP vaccine, improved and easy-to-use diagnostic assays for recurrent testing combined with isolation and treatment of positive animals represent an option for CBPP control in Africa. Here we describe the comprehensive screening of 17 immunogenic Mycoplasma mycoides subsp. mycoides proteins using well-characterized bovine sera for the development of a novel cocktail enzyme-linked immunosorbent assay (ELISA) for laboratory use. Two recombinant Mycoplasma immunogens, MSC_0136 and MSC_0636, were used to set up a standardized cocktail ELISA protocol. According to the results from more than 100 serum samples tested, the sensitivity and specificity of the novel cocktail ELISA were 85.6% and 96.4%, respectively, with an overall diagnostic accuracy comparable to that of the Office International des Epizooties (OIE)-prescribed serological assays. In addition, we provide a proof of principle for a field-applicable, easy-to-use commercially produced prototype lateral-flow test for rapid (<30-min) diagnosis of CBPP.

Camel Streptococcus agalactiae populations are associated with specific disease complexes and acquired the tetracycline resistance gene tetM via a Tn916-like element.

Camels are the most valuable livestock species in the Horn of Africa and play a pivotal role in the nutritional sustainability for millions of people. Their health status is therefore of utmost importance for the people living in this region. Streptococcus agalactiae, a Group B Streptococcus (GBS), is an important camel pathogen. Here we present the first epidemiological study based on genetic and phenotypic data from African camel derived GBS. Ninety-two GBS were characterized using multilocus sequence typing (MLST), capsular polysaccharide typing and in vitro antimicrobial susceptibility testing. We analysed the GBS using Bayesian linkage, phylogenetic and minimum spanning tree analyses and compared them with human GBS from East Africa in order to investigate the level of genetic exchange between GBS populations in the region. Camel GBS sequence types (STs) were distinct from other STs reported so far. We mapped specific STs and capsular types to major disease complexes caused by GBS. Widespread resistance (34%) to tetracycline was associated with acquisition of the tetM gene that is carried on a Tn916-like element, and observed primarily among GBS isolated from mastitis. The presence of tetM within different MLST clades suggests acquisition on multiple occasions. Wound infections and mastitis in camels associated with GBS are widespread and should ideally be treated with antimicrobials other than tetracycline in East Africa.

Mass loading, distribution, and removal of antibiotics and antiretroviral drugs in selected wastewater treatment plants in Kenya.

The discharge of active pharmaceutical ingredients (APIs) into the aquatic environment from wastewater effluents is a concern in many countries. Although many studies have been conducted to evaluate the APIs removal efficiencies and emissions to the environment in wastewater treatment plants (WWTPs), most of these studies considered the aqueous and sludge phases, disregarding the suspended particulate matter (SPM) phase. To try to understand the role of the SPM, the occurrence of five most common antibiotics and three antiretroviral drugs (ARVDs) commonly used in Kenya were investigated in this study. APIs partitioning and mass loading in influents and effluents of three different WWTPs: trickling filters, stabilization ponds, and decentralized fecal sludge system, were evaluated. API concentration levels ranging from ˂LOQ (limit of quantification) to 92 µgL-1 and ˂LOQ to 82.2 mgkg-1 were observed in aqueous samples and solid samples respectively, with SPM accounting for most of the higher concentrations. The use of the aqueous phase alone for determination of removal efficiencies showed underestimations of API removal as compared to when solid phases are also considered. Negative removal efficiencies were observed, depending on the compound and the type of WWTP. The negative removals were associated with deconjugation of metabolites, aggregated accumulation of APIs in the WWTPs, as well as unaccounted hydraulic retention time during sampling. Compound characteristics, environmental factors, and WWTPs operation influenced WWTPs removal efficiencies. Wastewater stabilization ponds had the poorest removals efficiencies with an average of -322%. High total mass loads into the WWTPs influent and effluent of 22,729 and 22,385 mg day-1 1000 PE-1 were observed respectively. The results aims at aiding scientists and engineers in planning and designing of WWTPs. Findings also aim at aiding policy-making on pharmaceutical drug use and recommend proper wastewater management practices to manage the high mass loading observed in the WWTPs.

Occurrence, distribution, and risk assessment of pharmerciuticals in wastewater and open surface drains of peri-urban areas: Case study of Juja town, Kenya.

The occurrence of Active Pharmaceutical Ingredients (APIs) in the environment is becoming a major area of concern due to their undesirable effects on non-target organisms. This study investigated the occurrence and risk of contamination by five antibiotics and three antiretrovirals drugs in a fast-growing peri-urban area in Kenya, with inadequate sewer system coverage. Due to poor sewage connectivity and poorly designed decentralized systems, wastewater is directly released in open drains. Water and sediment samples were collected from open surface water drains, while wastewater samples were collected from centralized wastewater treatment plants (WWTP). Solid-phase extraction and ultrasonic-assisted extraction for the aqueous and sediment samples respectively were carried out and extracts analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) using isotopically labeled internal standards. APIs were observed with the detection frequency ranging from 36% to 100%. High mean concentrations of 48.7 µg L-1, 108 µg L-1, and 532 µg L-1 were observed in surface drains for Lamivudine (3 TC), Sulfamethoxazole (SMX), Ciprofloxacin (CIP) respectively. Drain sediments also showed high concentrations of APIs ranging from 2.1 to 13,100 µg kg-1. APIs in this study exceeded those observed in existing literature studies. JKUAT WWTP removal efficiencies varied from -90.68% to 72.67%. Total APIs emission load of the study area was 3550 mg d-1 with WWTP effluent contributing higher loads (2620 mg d-1) than surface water drains (640 mg d-1). Zidovudine (ZDV), nevirapine (NVP), and trimethoprim (TMP) loads in drains, however, exceeded WWTP effluent. Low to high ecotoxicity risk of the individual APIs were observed to the aquatic environment, with high risks for the development of antibiotic resistance in microbiome as determined by the risk quotient (RQ) approach. Risk management through efficient wastewater collection, conveyance, and treatment is necessary to suppress the measured concentrations.

Assessment of in vitro interferon-gamma 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.

The origin of the 'Mycoplasma mycoides cluster' coincides with domestication of ruminants.

The 'Mycoplasma mycoides cluster' comprises the ruminant pathogens Mycoplasma mycoides subsp. mycoides the causative agent of contagious bovine pleuropneumonia (CBPP), Mycoplasma capricolum subsp. capripneumoniae the agent of contagious caprine pleuropneumonia (CCPP), Mycoplasma capricolum subsp. capricolum, Mycoplasma leachii and Mycoplasma mycoides subsp. capri. CBPP and CCPP are major livestock diseases and impact the agricultural sector especially in developing countries through reduced food-supply and international trade restrictions. In addition, these diseases are a threat to disease-free countries. We used a multilocus sequence typing (MLST) approach to gain insights into the demographic history of and phylogenetic relationships among the members of the 'M. mycoides cluster'. We collected partial sequences from seven housekeeping genes representing a total of 3,816 base pairs from 118 strains within this cluster, and five strains isolated from wild Caprinae. Strikingly, the origin of the 'M. mycoides cluster' dates to about 10,000 years ago, suggesting that the establishment and spread of the cluster coincided with livestock domestication. In addition, we show that hybridization and recombination may be important factors in the evolutionary history of the cluster.