Canine Staphylococcaceae circulating in a Kenyan animal shelter.

Animal shelters, especially in resource-poor countries, bring together pets from different regions and with different backgrounds. The crowding of such animals often results in infectious diseases, such as respiratory infections. This study characterized Staphylococcaceae from diseased and apparently healthy dogs housed in an animal shelter in Kenya, to determine their antibiotic resistance profiles, their genetic relatedness, and the presence of dominant clones. Therefore, bacteria were collected from all 167 dogs present in the shelter in June 2015 and screened for Staphylococcaceae using standard cultivation techniques. In all, 92 strains were isolated from 85 dogs and subsequently sequenced by PacBio long-read sequencing. Strains encompassed nine validated species, while S. aureus (n = 47), S. pseudintermedius (n = 21), and Mammaliicoccus (M.) sciuri (n = 16) were the three most dominant species. Two S. aureus clones of ST15 (CC15) and ST1292 (CC1) were isolated from 7 and 37 dogs, respectively. All 92 strains isolated were tested for their antimicrobial susceptibility by determining the minimum inhibitory concentrations. In all, 86 strains had resistance-associated minimal inhibitory concentrations to at least one of the following antimicrobials: tetracycline, benzylpenicillin, oxacillin, erythromycin, clindamycin, trimethoprim, kanamycin/gentamicin, or streptomycin. Many virulence-encoding genes were detected in the S. aureus strains, other Staphylococcaceae contained a different set of homologs of such genes. The presence of mobile genetic elements, such as plasmids and prophages, known to facilitate the dissemination of virulence- and resistance-encoding genes, was also assessed. The unsuspected high presence of two S. aureus clones in about 50% of dogs suggests dissemination within the shelter and a human source.IMPORTANCEMicrobiological data from sub-Saharan Africa are scarce compared to data from North America, Europe, or Asia, and data derived from dogs, the man's best friend, kept in sub-Saharan Africa are largely missing. This work presents data on Staphylococcaceae mainly isolated from the nasal cavity of dogs stationed at a Kenyan shelter in 2015. We characterized 92 strains isolated from 85 dogs, diseased and apparently healthy ones. The strains isolated covered nine validated species and we determined their phenotypic resistance and characterized their complete genomes. Interestingly, Staphylococcus aureus of two predominant genetic lineages, likely to be acquired from humans, colonized many dogs. We also detected 15 novel sequence types of Mammaliicoccus sciuri and S. pseudintermedius indicating sub-Saharan-specific phylogenetic lineages. The data presented are baseline data that guide antimicrobial treatment for dogs in the region.

Genomic Characterization and Antimicrobial Susceptibility of Dromedary-Associated Staphylococcaceae from the Horn of Africa.

Members of the Staphylococcaceae family, particularly those of the genus Staphylococcus, encompass important human and animal pathogens. We collected and characterized Staphylococcaceae strains from apparently healthy and diseased camels (n = 84) and cattle (n = 7) in Somalia and Kenya. We phenotypically characterized the strains, including their antimicrobial inhibitory concentrations. Then, we sequenced their genomes using long-read sequencing, closed their genomes, and subsequently compared and mapped their virulence- and resistance-associated gene pools. Genome-based phylogenetics revealed 13 known Staphylococcaceae and at least two novel species. East African strains of different species encompassed novel sequence types and phylogenetically distant clades. About one-third of the strains had non-wild-type MICs. They were resistant to at least one of the following antimicrobials: tetracycline, benzylpenicillin, oxacillin, erythromycin, clindamycin, trimethoprim, gentamicin, or streptomycin, encoded by tet(K), blaZ/blaARL, mecA/mecA1, msrA/mphC, salA, dfrG, aacA-aphD, and str, respectively. We identified the first methicillin- and multidrug-resistant camel S. epidermidis strain of sequence type (ST) 1136 in East Africa. The pool of virulence-encoding genes was largest in the S. aureus strains, as expected, although other rather commensal strains contained distinct virulence-encoding genes. We identified toxin-antitoxin (TA) systems such as the hicA/hicB and abiEii/abiEi families, reported here for the first time for certain species of Staphylococcaceae. All strains contained at least one intact prophage sequence, mainly belonging to the Siphoviridae family. We pinpointed potential horizontal gene transfers between camel and cattle strains and also across distinct Staphylococcaceae clades and species. IMPORTANCE Camels are a high value and crucial livestock species in arid and semiarid regions of Africa and gain importance giving the impact of climate change on traditional livestock species. Our current knowledge with respect to Staphylococcaceae infecting camels is very limited compared to that for other livestock species. Better knowledge will foster the development of specific diagnostic assays, guide promising antimicrobial treatment options, and inform about potential zoonotic risks. We characterized 84 Staphylococcaceae strains isolated from camels with respect to their antimicrobial resistance and virulence traits. We detected potentially novel Staphylococcus species, resistances to different classes of antimicrobials, and the first camel multidrug-resistant S. epidermidis strain of sequence type 1136.

Reproduction of contagious bovine pleuropneumonia via aerosol-based challenge with Mycoplasma mycoides subsp. mycoides.

Contagious bovine pleuropneumonia (CBPP) is a respiratory disease caused by Mycoplasma mycoides subsp. mycoides. Infection occurs via Mycoplasma-containing droplets and therefore requires close contact between animals. The current infection models are suboptimal and based on intratracheal installation of mycoplasmas or in-contact infection. This work tested the infection of adult cattle via aerosols containing live mycoplasmas mimicking the infection of cattle in the field. Therefore, we infected six cattle with aerosolized Mycoplasma mycoides subsp. mycoides strain Afadé over seven consecutive days with altogether 109 colony forming units. All animals seroconverted between 11-24 days post infection and five out of six animals showed typical CBPP lesions. One animal did not show any lung lesions at necropsy, while another animal had to be euthanized at 25 days post infection because it reached endpoint criteria. Seroconversion confirmed successful infection and the spectrum of clinical and lesions observed mirrors epidemiological models and the field situation, in which only a fraction of animals suffers from acute clinical disease post infection.

Hepatitis E Virus Genotype 7 RNA and Antibody Kinetics in Naturally Infected Dromedary Calves, United Arab Emirates.

Orthohepevirus A genotype 7 is a novel zoonotic variant of hepatitis E virus. To clarify infection in the animal reservoir, we virologically monitored 11 dromedary dam-calf pairs. All calves became infected during the first 6 months of life and cleared the virus after an average of 2 months. Dams did not become infected.

Contagious Bovine and Caprine Pleuropneumonia: a research community's recommendations for the development of better vaccines.

Contagious bovine pleuropneumonia (CBPP) and contagious caprine pleuropneumonia (CCPP) are major infectious diseases of ruminants caused by mycoplasmas in Africa and Asia. In contrast with the limited pathology in the respiratory tract of humans infected with mycoplasmas, CBPP and CCPP are devastating diseases associated with high morbidity and mortality. Beyond their obvious impact on animal health, CBPP and CCPP negatively impact the livelihood and wellbeing of a substantial proportion of livestock-dependent people affecting their culture, economy, trade and nutrition. The causative agents of CBPP and CCPP are Mycoplasma mycoides subspecies mycoides and Mycoplasma capricolum subspecies capripneumoniae, respectively, which have been eradicated in most of the developed world. The current vaccines used for disease control consist of a live attenuated CBPP vaccine and a bacterin vaccine for CCPP, which were developed in the 1960s and 1980s, respectively. Both of these vaccines have many limitations, so better vaccines are urgently needed to improve disease control. In this article the research community prioritized biomedical research needs related to challenge models, rational vaccine design and protective immune responses. Therefore, we scrutinized the current vaccines as well as the challenge-, pathogenicity- and immunity models. We highlight research gaps and provide recommendations towards developing safer and more efficacious vaccines against CBPP and CCPP.

Antimicrobial resistant and extended-spectrum ß-lactamase (ESBL) producing Escherichia coli isolated from fecal samples of African dromedary camels.

This study was conducted to determine the distribution of antimicrobial resistance among Escherichia coli isolated from feces of healthy dromedary camels in Kenya. A total of 162 fecal samples were cultivated for E. coli. Samples were also subcultivated to detect E. coli with extended-spectrum ß-lactamases (ESBLs). Antimicrobial susceptibility testing (AST) was performed by disk diffusion using a panel of 16 antimicrobials. In addition, isolates were screened for the presence of the plasmid-mediated colistin resistance genes mcr-1 to mcr-5. Samples from 20 (12.4%) of the camels contained antimicrobial resistant (AMR) E. coli, and 85% of the AMR isolates were multidrug resistant (MDR). The highest frequency of resistance was observed to tetracycline (11.7%), followed by ampicillin and streptomycin (both 10.5%), and sulfamethoxazole/trimethoprim (9.9%). Two (1.2%) of the isolates showed intermediate resistance to cefazolin and streptomycin, respectively. All the isolates were susceptible to amoxycillin/clavulanic acid, ciprofloxacin, fosfomycin, aztreonam and kanamycin, and 86.4% of the isolates were susceptible to all 16 antimicrobials used in this study. The prevalence of fecal carriage of ESBL producing E. coli was 0.6%. PCR and amplicon sequencing showed that the ESBL producer belonged to E. coli phylogenetic group A, sequence type (ST) 48, and harbored bla CTX-M-15. None of the isolates contained mcr genes. The results indicate that dromedary camels in Kenya may be reservoirs of AMR E. coli, including ESBL producers, that could potentially be transmitted to humans by direct contact or via the food chain.

Multiplicity of Asymptomatic Plasmodium falciparum Infections and Risk of Clinical Malaria: A Systematic Review and Pooled Analysis of Individual Participant Data.

BACKGROUND: The malaria parasite Plasmodium falciparum holds an extensive genetic polymorphism. In this pooled analysis, we investigate how the multiplicity in asymptomatic P. falciparum infections-that is, the number of coinfecting clones-affects the subsequent risk of clinical malaria in populations living under different levels of transmission. METHODS: A systematic search of the literature was performed to identify studies in which P. falciparum infections were genotyped in asymptomatic individuals who were followed up prospectively regarding the incidence of clinical malaria. Individual participant data were pooled from 15 studies (n = 3736 individuals). RESULTS: Multiclonal asymptomatic infections were associated with a somewhat increased subsequent risk of clinical malaria in the youngest children, followed by an initial declining risk with age irrespective of transmission intensity. At approximately 5 years of age, the risk continued the gradual decline with age in high-transmission settings. However, in older children in moderate-, low-, and seasonal-transmission settings, multiclonal infections were either not significantly associated with the risk of subsequent febrile malaria or were associated with an increased risk. CONCLUSIONS: The number of clones in asymptomatic P. falciparum infections is associated with different risks of subsequent clinical malaria depending on age and transmission intensity.

Attenuation of a Pathogenic Mycoplasma Strain by Modification of the obg Gene by Using Synthetic Biology Approaches.

Mycoplasma species are responsible for several economically significant livestock diseases for which there is a need for new and improved vaccines. Most of the existing mycoplasma vaccines are attenuated strains that have been empirically obtained by serial passages or by chemical mutagenesis. The recent development of synthetic biology approaches has opened the way for the engineering of live mycoplasma vaccines. Using these tools, the essential GTPase-encoding gene obg was modified directly on the Mycoplasma mycoides subsp. capri genome cloned in yeast, reproducing mutations suspected to induce a temperature-sensitive (TS+) phenotype. After transplantation of modified genomes into a recipient cell, the phenotype of the resulting M. mycoides subsp. capri mutants was characterized. Single-point obg mutations did not result in a strong TS+ phenotype in M. mycoides subsp. capri, but a clone presenting three obg mutations was shown to grow with difficulty at temperatures of ≥40°C. This particular mutant was then tested in a caprine septicemia model of M. mycoides subsp. capri infection. Five out of eight goats infected with the parental strain had to be euthanized, in contrast to one out of eight goats infected with the obg mutant, demonstrating an attenuation of virulence in the mutant. Moreover, the strain isolated from the euthanized animal in the group infected with the obg mutant was shown to carry a reversion in the obg gene associated with the loss of the TS+ phenotype. This study demonstrates the feasibility of building attenuated strains of mycoplasma that could contribute to the design of novel vaccines with improved safety.IMPORTANCE Animal diseases due to mycoplasmas are a major cause of morbidity and mortality associated with economic losses for farmers all over the world. Currently used mycoplasma vaccines exhibit several drawbacks, including low efficacy, short time of protection, adverse reactions, and difficulty in differentiating infected from vaccinated animals. Therefore, there is a need for improved vaccines to control animal mycoplasmoses. Here, we used genome engineering tools derived from synthetic biology approaches to produce targeted mutations in the essential GTPase-encoding obg gene of Mycoplasma mycoides subsp. capri Some of the resulting mutants exhibited a marked temperature-sensitive phenotype. The virulence of one of the obg mutants was evaluated in a caprine septicemia model and found to be strongly reduced. Although the obg mutant reverted to a virulent phenotype in one infected animal, we believe that these results contribute to a strategy that should help in building new vaccines against animal mycoplasmoses.

Removal of a Subset of Non-essential Genes Fully Attenuates a Highly Virulent Mycoplasma Strain.

Mycoplasmas are the smallest free-living organisms and cause a number of economically important diseases affecting humans, animals, insects, and plants. Here, we demonstrate that highly virulent Mycoplasma mycoides subspecies capri (Mmc) can be fully attenuated via targeted deletion of non-essential genes encoding, among others, potential virulence traits. Five genomic regions, representing approximately 10% of the original Mmc genome, were successively deleted using Saccharomyces cerevisiae as an engineering platform. Specifically, a total of 68 genes out of the 432 genes verified to be individually non-essential in the JCVI-Syn3.0 minimal cell, were excised from the genome. In vitro characterization showed that this mutant was similar to its parental strain in terms of its doubling time, even though 10% of the genome content were removed. A novel in vivo challenge model in goats revealed that the wild-type parental strain caused marked necrotizing inflammation at the site of inoculation, septicemia and all animals reached endpoint criteria within 6 days after experimental infection. This is in contrast to the mutant strain, which caused no clinical signs nor pathomorphological lesions. These results highlight, for the first time, the rational design, construction and complete attenuation of a Mycoplasma strain via synthetic genomics tools. Trait addition using the yeast-based genome engineering platform and subsequent in vitro or in vivo trials employing the Mycoplasma chassis will allow us to dissect the role of individual candidate Mycoplasma virulence factors and lead the way for the development of an attenuated designer vaccine.

Shiga toxin-producing Escherichia coli (STEC) isolated from fecal samples of African dromedary camels.

Shiga toxin-producing Escherichia coli (STEC) cause gastrointestinal illnesses including non-bloody or bloody diarrhoea, haemorrhagic colitis (HC), and the haemolytic uremic syndrome (HUS). To investigate the occurrence of STEC among grazing dromedaries from Kenya, E. coli isolated from fecal matter collected from 163 dromedaries on a large ranch were screened for the presence of stx1 and stx2. STEC strains were isolated and serotyped. Isolates were subjected to PCR for the subtyping of stx genes and for the detection of eae and ehx. In addition, whole genome sequencing (WGS) was carried out to detect further virulence genes and to determine the multilocus sequence types (MLST). Antimicrobial resistance profiles were determined by disk diffusion. STEC was isolated from 20 (12.3%) of the fecal samples. Thereof, nine (45%) isolates were STEC O156:H25, three (15%) isolates typed STEC O43:H2. The remaining isolates occurred as single serotypes or were O non-typeable. Eleven (55%) of the isolates harboured stx2a, nine (45%) eae, and 14 (70%) ehx, respectively. WGS revealed the presence of iss in 16 (80%), subAB in four (20%) and astA in two (10%) of the isolates, Furthermore, espA, tccP, nleA, nleB, tccP, and tir were found exclusively among STEC O156:H25. Eleven different sequence types (ST) were detected. The most prominent was ST300/ST5343, which comprised STEC O156:H25. All STEC isolates were pan susceptible to a panel of 16 antimicrobial agents. Overall, the results indicate that dromedary camels in Kenya may be reservoirs of STEC, including serotypes possessing virulence markers associated to disease in humans, such as STEC O156:H25. STEC in camels may represent a health hazard for humans with close contact to camels or to consumers of camel derived foodstuffs, such as unpasteurised camel milk.

Reproduction of contagious caprine pleuropneumonia reveals the ability of convalescent sera to reduce hydrogen peroxide production in vitro.

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.

In vivo role of capsular polysaccharide in Mycoplasma mycoides.

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.

Link of a ubiquitous human coronavirus to dromedary camels.

The four human coronaviruses (HCoVs) are globally endemic respiratory pathogens. The Middle East respiratory syndrome (MERS) coronavirus (CoV) is an emerging CoV with a known zoonotic source in dromedary camels. Little is known about the origins of endemic HCoVs. Studying these viruses' evolutionary history could provide important insight into CoV emergence. In tests of MERS-CoV-infected dromedaries, we found viruses related to an HCoV, known as HCoV-229E, in 5.6% of 1,033 animals. Human- and dromedary-derived viruses are each monophyletic, suggesting ecological isolation. One gene of dromedary viruses exists in two versions in camels, full length and deleted, whereas only the deleted version exists in humans. The deletion increased in size over a succession starting from camelid viruses via old human viruses to contemporary human viruses. Live isolates of dromedary 229E viruses were obtained and studied to assess human infection risks. The viruses used the human entry receptor aminopeptidase N and replicated in human hepatoma cells, suggesting a principal ability to cause human infections. However, inefficient replication in several mucosa-derived cell lines and airway epithelial cultures suggested lack of adaptation to the human host. Dromedary viruses were as sensitive to the human type I interferon response as HCoV-229E. Antibodies in human sera neutralized dromedary-derived viruses, suggesting population immunity against dromedary viruses. Although no current epidemic risk seems to emanate from these viruses, evolutionary inference suggests that the endemic human virus HCoV-229E may constitute a descendant of camelid-associated viruses. HCoV-229E evolution provides a scenario for MERS-CoV emergence.

Hepatitis E Virus Infection in Dromedaries, North and East Africa, United Arab Emirates, and Pakistan, 1983-2015.

A new hepatitis E virus (HEV-7) was recently found in dromedaries and 1 human from the United Arab Emirates. We screened 2,438 dromedary samples from Pakistan, the United Arab Emirates, and 4 African countries. HEV-7 is long established, diversified and geographically widespread. Dromedaries may constitute a neglected source of zoonotic HEV infections.

MERS-CoV Antibodies in Humans, Africa, 2013-2014.

Dromedaries in Africa and elsewhere carry the Middle East respiratory syndrome coronavirus (MERS-CoV). To search for evidence of autochthonous MERS-CoV infection in humans, we tested archived serum from livestock handlers in Kenya for MERS-CoV antibodies. Serologic evidence of infection was confirmed for 2 persons sampled in 2013 and 2014.

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.

Recombinant Mycoplasma mycoides proteins elicit protective immune responses against contagious bovine pleuropneumonia.

Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a devastating respiratory disease mainly affecting cattle in sub-Saharan Africa. The current vaccines are based on live-attenuated Mmm strains and present problems with temperature stability, duration of immunity and adverse reactions, thus new vaccines are needed to overcome these issues. We used a reverse vaccinology approach to identify 66 Mmm potential vaccine candidates. The selection and grouping of the antigens was based on the presence of specific antibodies in sera from CBPP-positive animals. The antigens were used to immunize male Boran cattle (Bos indicus) followed by a challenge with the Mmm strain Afadé. Two of the groups immunized with five proteins each showed protection after the Mmm challenge (Groups A and C; P<0.05) and in one group (Group C) Mmm could not be cultured from lung specimens. A third group (Group N) showed a reduced number of animals with lesions and the cultures for Mmm were also negative. While immunization with some of the antigens conferred protection, others may have increased immune-related pathology. This is the first report that Mmm recombinant proteins have been successfully used to formulate a prototype vaccine and these results pave the way for the development of a novel commercial vaccine.

Morphological characterization and immunohistochemical detection of the proinflammatory cytokines IL-1ß, IL-17A, and TNF-α in lung lesions associated with contagious bovine pleuropneumonia.

Contagious bovine pleuropneumonia (CBPP), a severe respiratory disease, is characterized by massive inflammation of the lung especially during the acute clinical stage of infection. Tissue samples from cattle, experimentally infected with Mycoplasma mycoides subsp. mycoides Afadé, were subjected to histopathological and immunohistochemical examination in order to provide insight into innate immune pathways that shape inflammatory host responses. Lung lesions were characterized by vasculitis, necrosis, and increased presence of macrophages and neutrophils, relative to uninfected animals. The presence of three cytokines associated with innate inflammatory immune responses, namely, IL-1ß, IL-17A, and TNF-α, were qualitatively investigated in situ. Higher cytokine levels were detected in lung tissue samples from CBPP-affected cattle compared to samples derived from an uninfected control group. We therefore conclude that the cytokines TNF-α and IL-1ß, which are prevalent in the acute phase of infections, play a role in the inflammatory response seen in the lung tissue in CBPP. IL-17A gets released by activated macrophages and attracts granulocytes that modulate the acute phase of the CBPP lesions.

Proteomic characterization of pleural effusion, a specific host niche of Mycoplasma mycoides subsp. mycoides from cattle with contagious bovine pleuropneumonia (CBPP).

Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a severe pleuropneumonia in cattle. The abnormal accumulation of pleural fluid, called pleural effusion (PE), is one of the characteristics of this disease. We performed a proteomic analysis of seven PE samples from experimentally infected cattle and characterized their composition with respect to bovine and Mmm proteins. We detected a total of 963 different bovine proteins. Further analysis indicated a strong enrichment of proteins involved in antigen processing, platelet activation and degranulation and apoptosis and an increased abundance of acute phase proteins.With regard to the pathogen, up to 108 viable mycoplasma cells per ml were detected in the PE supernatant. The proteomic analysis revealed 350 mycoplasma proteins, including proteins involved in virulence-associated processes like hydrogen peroxide (H2O2) production and capsule synthesis. The bovine proteins detected will aid to characterize the inflammasome during an acute pleuropneumonia in cattle and the identified mycoplasma proteins will serve as baseline data to be compared with in vitro studies to improve our understanding of pathogenicity mechanisms. Based on our results, we named the pleural effusion an "in vivo niche" of Mmm during the acute phase of CBPP. Biological significance: This is the first study on bovine pleural effusions derived from an infectious disease and the first approach to characterize the proteome of Mycoplasma mycoides in vivo. This study revealed a high number of viable Mmm cells in the pleural effusion. The bovine pleural effusion proteome during Mmm infection is qualitatively similar to plasma, but differs with respect to high abundance of acute phase proteins. On the other hand,Mmm in its natural host produces proteins involved in capsule synthesis, H2O2 production and induction of inflammatory response, supporting previous knowledge on mechanisms underlying the survival and virulence of this pathogen while inside the natural host. This knowledge forms a profound basis for testing the identified protein candidates for diagnostics or vaccines.

Complete genome sequence of Staphylococcus aureus, strain ILRI_Eymole1/1, isolated from a Kenyan dromedary camel.

We report the genome of a Staphylococcus aureus strain (ILRI_Eymole1/1) isolated from a nasal swab of a dromedary camel (Camelus dromedarius) in North Kenya. The complete genome sequence of this strain consists of a circular chromosome of 2,874,302 bp with a GC-content of 32.88 %. In silico annotation predicted 2755 protein-encoding genes and 76 non-coding genes. This isolate belongs to MLST sequence type 30 (ST30). Phylogenetic analysis based on a subset of 283 core genes revealed that it falls within the human clonal complex 30 (CC30) S. aureus isolate cluster but is genetically distinct. About 79 % of the protein encoding genes are part of the CC30 core genome (genes common to all CC30 S. aureus isolates), ~18 % were within the variable genome (shared among multiple but not all isolates) and ~ 3 % were found only in the genome of the camel isolate. Among the 85 isolate-specific genes, 79 were located within putative phages and pathogenicity islands. Protein encoding genes associated with bacterial adhesion, and secretory proteins that are essential components of the type VII secretion system were also identified. The complete genome sequence of S. aureus strain ILRI_Eymole1/1 has been deposited in the European Nucleotide Archive under the accession no LN626917.1.