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.

Ehrlichia spp. close to Ehrlichia ruminantium, Ehrlichia canis, and "Candidatus Ehrlichia regneryi" linked to heartwater-like disease in Kenyan camels (Camelus dromedarius).

We present findings from an outbreak of a heartwater-like disease in camels that killed at least 2000 adult animals in Kenya in 2016. Clinical signs included excitability, head pressing, aimless wandering, recumbency, and fast breathing followed by death after about 4 days. The observed morbidity in one herd was 40% with an average mortality of 7.5% in animals that received early antibiotic treatments. In untreated adults, the case fatality rate reached 100%. Gross pathology showed pulmonary edema, pleural exudate, hydrothorax, hydropericardium, ascites, enlarged "cooked" liver, nephrosis, and blood in the abomasum and intestine. Using established PCR-based protocols for tick-borne pathogens, a sequence close to Ehrlichia regneryi and Ehrlichia canis amplified in blood from two sick camels. We also amplified an Ehrlichia sp. sequence close to Ehrlichia ruminantium Welgevonden from a pool of Amblyomma spp. ticks collected from a sick camel and in a pool of Rhipicephalus spp. ticks from healthy camels.

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.

Molecular study on Pasteurella multocida and Mannheimia granulomatis from Kenyan Camels (Camelus dromedarius).

BACKGROUND: Outbreaks of a Haemorrhagic Septicaemia (HS) like disease causing large mortalities in camels (Camelus dromedarius) in Asia and in Africa have been reported since 1890. Yet the aetiology of this condition remains elusive. This study is the first to apply state of the art molecular methods to shed light on the nasopharyngeal carrier state of Pasteurellaceae in camels. The study focused on HS causing Pasteurella multocida capsular types B and E. Other Pasteurellaceae, implicated in common respiratory infections of animals, were also investigated. METHODS: In 2007 and 2008, 388 nasopharyngeal swabs were collected at 12 locations in North Kenya from 246 clinically healthy camels in 81 herds that had been affected by HS-like disease. Swabs were used to cultivate bacteria on blood agar and to extract DNA for subsequent PCR analysis targeting P. multocida and Mannheimia-specific gene sequences. RESULTS: Forty-five samples were positive for P. multocida genes kmt and psl and for the P. multocida Haemorrhagic Septicaemia (HS) specific sequences KTSP61/KTT72 but lacked HS-associated capsular type B and E genes capB and capE. This indicates circulation of HS strains in camels that lack established capsular types. Sequence analysis of the partial 16S rRNA gene identified 17 nasal swab isolates as 99% identical with Mannheimia granulomatis, demonstrating a hitherto unrecognised active carrier state for M. granulomatis or a closely related Mannheimia sp. in camels. CONCLUSIONS: The findings of this study provide evidence for the presence of acapsular P. multocida or of hitherto unknown capsular types of P. multocida in camels, closely related to P. multocida strains causing HS in bovines. Further isolations and molecular studies of camelid P. multocida from healthy carriers and from HS-like disease in camels are necessary to provide conclusive answers. This paper is the first report on the isolation of M. granulomatis or a closely related new Mannheimia species from camelids.

East and West African milk products are reservoirs for human and livestock-associated Staphylococcus aureus.

Staphylococcus aureus frequently isolated from milk products in sub-Saharan Africa (SSA) is a major pathogen responsible for food intoxication, human and animal diseases. SSA hospital-derived strains are well studied but data on the population structure of foodborne S. aureus required to identify possible staphylococcal food poisoning sources is lacking. Therefore, the aim was to assess the population genetic structure, virulence and antibiotic resistance genes associated with milk-derived S. aureus isolates from Côte d'Ivoire, Kenya and Somalia through spa-typing, MLST, and DNA microarray analysis. Seventy milk S. aureus isolates from the three countries were assigned to 27 spa (7 new) and 23 (12 new) MLST sequence types. Milk-associated S. aureus of the three countries is genetically diverse comprising human and livestock-associated clonal complexes (CCs) predominated by the CC5 (n = 10) and CC30 (n = 9) isolates. Panton-Valentine leukocidin, toxic shock syndrome toxin and enterotoxin encoding genes were predominantly observed among human-associated CCs. Penicillin, fosfomycin and tetracycline, but not methicillin resistance genes were frequently detected. Our findings indicate that milk-associated S. aureus in SSA originates from human and animal sources alike highlighting the need for an overarching One Health approach to reduce S. aureus disease burdens through improving production processes, animal care and hygienic measures.

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.

Field-Applicable Recombinase Polymerase Amplification Assay for Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae.

Contagious caprine pleuropneumonia (CCPP) is a highly contagious disease caused by Mycoplasma capricolum subsp. capripneumoniae that affects goats in Africa and Asia. Current available methods for the diagnosis of Mycoplasma infection, including cultivation, serological assays, and PCR, are time-consuming and require fully equipped stationary laboratories, which make them incompatible with testing in the resource-poor settings that are most relevant to this disease. We report a rapid, specific, and sensitive assay employing isothermal DNA amplification using recombinase polymerase amplification (RPA) for the detection of M. capricolum subsp. capripneumoniae. We developed the assay using a specific target sequence in M. capricolum subsp. capripneumoniae, as found in the genome sequence of the field strain ILRI181 and the type strain F38 and that was further evidenced in 10 field strains from different geographical regions. Detection limits corresponding to 5 × 10(3) and 5 × 10(4) cells/ml were obtained using genomic DNA and bacterial culture from M. capricolum subsp. capripneumoniae strain ILRI181, while no amplification was obtained from 71 related Mycoplasma isolates or from the Acholeplasma or the Pasteurella isolates, demonstrating a high degree of specificity. The assay produces a fluorescent signal within 15 to 20 min and worked well using pleural fluid obtained directly from CCPP-positive animals without prior DNA extraction. We demonstrate that the diagnosis of CCPP can be achieved, with a short sample preparation time and a simple read-out device that can be powered by a car battery, in <45 min in a simulated field setting.

MERS coronavirus neutralizing antibodies in camels, Eastern Africa, 1983-1997.

To analyze the distribution of Middle East respiratory syndrome coronavirus (MERS-CoV)-seropositive dromedary camels in eastern Africa, we tested 189 archived serum samples accumulated during the past 30 years. We identified MERS-CoV neutralizing antibodies in 81.0% of samples from the main camel-exporting countries, Sudan and Somalia, suggesting long-term virus circulation in these animals.

Antibodies against MERS coronavirus in dromedary camels, Kenya, 1992-2013.

Dromedary camels are a putative source for human infections with Middle East respiratory syndrome coronavirus. We showed that camels sampled in different regions in Kenya during 1992-2013 have antibodies against this virus. High densities of camel populations correlated with increased seropositivity and might be a factor in predicting long-term virus maintenance.

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.

Detection of Antibodies to Ehrlichia spp. in Dromedary Camels and Co-Grazing Sheep in Northern Kenya Using an Ehrlichia ruminantium Polyclonal Competitive ELISA.

A disease with clinical and post-mortem presentation similar to those seen in heartwater, a tick-borne disease of domestic and wild ruminants caused by the intracellular bacterium Ehrlichia ruminantium, was first reported in dromedary camels in Kenya in 2016; investigations carried out at the time to determine the cause were inconclusive. In the present study, we screened sera from Kenyan camels collected before (2015) and after (2020) the 2016 disease outbreak for antibodies to Ehrlichia spp. using an E. ruminantium polyclonal competitive ELISA (PC-ELISA). Median antibody levels were significantly higher (p < 0.0001) amongst camels originating from areas where the heartwater-like disease was reported than from disease-free areas, for animals sampled in both 2015 and 2020. Overall median seropositivity was higher in camels sampled in 2015 than in 2020, which could have been due to higher mean age in the former group. Camels that were PCR-positive for Candidatus Ehrlichia regneryi had significantly lower (p = 0.03) median antibody levels than PCR-negative camels. Our results indicate that Kenyan camels are frequently exposed to E. ruminantium from an early age, E. ruminantium was unlikely to have been the sole cause of the outbreak of heartwater-like disease; and Ca. E. regneryi does not appreciably cross-react with E. ruminantium in the PC-ELISA.

Ticks and Tick-Borne Pathogens Associated with Dromedary Camels (Camelus dromedarius) in Northern Kenya.

Ticks and tick-borne pathogens (TBPs) are major constraints to camel health and production, yet epidemiological data on their diversity and impact on dromedary camels remain limited. We surveyed the diversity of ticks and TBPs associated with camels and co-grazing sheep at 12 sites in Marsabit County, northern Kenya. We screened blood and ticks (858 pools) from 296 camels and 77 sheep for bacterial and protozoan TBPs by high-resolution melting analysis and sequencing of PCR products. Hyalomma (75.7%), Amblyomma (17.6%) and Rhipicephalus (6.7%) spp. ticks were morphologically identified and confirmed by molecular analyses. We detected TBP DNA in 80.1% of blood samples from 296 healthy camels. "Candidatus Anaplasma camelii", "Candidatus Ehrlichia regneryi" and Coxiella burnetii were detected in both camels and associated ticks, and Ehrlichia chaffeensis, Rickettsia africae, Rickettsia aeschlimannii and Coxiella endosymbionts were detected in camel ticks. We also detected Ehrlichia ruminantium, which is responsible for heartwater disease in ruminants, in Amblyomma ticks infesting camels and sheep and in sheep blood, indicating its endemicity in Marsabit. Our findings also suggest that camels and/or the ticks infesting them are disease reservoirs of zoonotic Q fever (C. burnetii), ehrlichiosis (E. chaffeensis) and rickettsiosis (R. africae), which pose public health threats to pastoralist communities.

Draft Genome Sequences of Seven Streptococcus agalactiae Strains Isolated from Camelus dromedarius at the Horn of Africa.

We present draft whole-genome sequences of seven Streptococcus agalactiae strains isolated from Camelus dromedarius in Kenya and Somalia. These data are an extension to the group B Streptococcus (GBS) pangenome and might provide more insight into the underlying mechanisms of pathogenicity and antibiotic resistance of camel GBS.

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.

Genome Sequences of Two Pathogenic Streptococcus agalactiae Isolates from the One-Humped Camel Camelus dromedarius.

Streptococcus agalactiae causes a range of clinical syndromes in camels (Camelus dromedarius). We report the genome sequences of two S. agalactiae isolates that induce abscesses in Kenyan camels. These genomes provide novel data on the composition of the S. agalactiae "pan genome" and reveal the presence of multiple genomic islands.