Anne Liljander

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.

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.

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

ABSTRACT 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 × 103 and 5 × 104 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-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.

Characterization of the in vitro core surface proteome of Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia §

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides (Mmm) is a severe cattle disease, present in many countries in sub-Saharan Africa. The development of improved diagnostic tests and vaccines for CBPP control remains a research priority. Polyacrylamide gel electrophoresis and mass spectrometry were used to characterize the Triton X-114 soluble proteome of nine Mmm strains isolated from Europe or Africa. Of a total of 250 proteins detected, 67 were present in all strains investigated. Of these, 44 were predicted to be lipoproteins or cytoplasmic membrane-associated proteins and are thus likely to be members of the core in vitro surface membrane-associated proteome of Mmm. Moreover, the presence of all identified proteins in other ruminant Mycoplasma pathogens were investigated. Two proteins of the core proteome were identified only in other cattle pathogens of the genus Mycoplasma pointing towards a role in host-pathogen interactions. The data generated will facilitate the identification and prioritization of candidate Mycoplasma antigens for improved control measures, as it is likely that surface-exposed membrane proteins will include those that are involved in host-pathogen interactions.

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.

Link of a ubiquitous human coronavirus to dromedary camels

Significance Our results raise a scenario for the natural history of a ubiquitous respiratory coronavirus (CoV) that has established itself in humans after it was likely acquired from camels. This scenario reminds us of the pandemic potential of the Middle East respiratory syndrome CoV, an agent that is thought to be acquired from camels without presently causing sustained human-to-human transmission. 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.

High antibody titres against predicted Mycoplasma surface proteins do not prevent sequestration in infected lung tissue in the course of experimental contagious bovine pleuropneumonia

Contagious bovine pleuropneumonia (CBPP), a severe respiratory disease of cattle caused by Mycoplasma mycoides subsp. mycoides (Mmm) is endemic in many African countries due to fragmented veterinary services and the lack of an efficient vaccine and sensitive diagnostics. More efficient tools to control the disease are needed, but to develop the tools, a better understanding of host-pathogen interactions is necessary. The aim of this study was to characterize the kinetics of the humoral immune response against 65 Mmm surface antigens for an extended period in cattle that survived a primary infection with Mmm. We describe clinical and haematological outcomes, and dissect the humoral immune response over time, to specific antigens and compared the antibody responses between different pathomorphological outcomes. No antigen-specific antibodies correlating with protection were identified. Interestingly we found that animals that developed Mycoplasma-containing sequestra had significantly higher antibody levels against proteins comprising the surface proteome than the animals that cleared Mycoplasma from their lungs. Based on these data we suggest that high antibody titres might play a role in the establishment of pathomorphological changes, such as vasculitis, which should be investigated in future studies. Beneficial antibody specificities and cellular immune responses need to be identified in order to foster the development of an improved vaccine in the future.

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.

Analysis of immune responses to recombinant proteins from strains of Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia.

Current contagious bovine pleuropneumonia (CBPP) vaccines are based on live-attenuated strains of Mycoplasma mycoides subsp. mycoides (Mmm). These vaccines have shortcomings in terms of efficacy, duration of immunity and in some cases show severe side effects at the inoculation site; hence the need to develop new vaccines to combat the disease. Reverse vaccinology approaches were used and identified 66 candidate Mycoplasma proteins using available Mmm genome data. These proteins were ranked by their ability to be recognized by serum from CBPP-positive cattle and thereafter used to inoculate naïve cattle. We report here the inoculation of cattle with recombinant proteins and the subsequent humoral and T-cell-mediated immune responses to these proteins and conclude that a subset of these proteins are candidate molecules for recombinant protein-based subunit vaccines for CBPP control.