A toolbox for manipulating the genome of the major goat pathogen, Mycoplasma capricolum subsp. capripneumoniae.

Mycoplasma capricolum subspecies capripneumoniae (Mccp) is the causative agent of contagious caprine pleuropneumonia (CCPP), a devastating disease listed by the World Organisation for Animal Health (WOAH) as a notifiable disease and threatening goat production in Africa and Asia. Although a few commercial inactivated vaccines are available, they do not comply with WOAH standards and there are serious doubts regarding their efficacy. One of the limiting factors to comprehend the molecular pathogenesis of CCPP and develop improved vaccines has been the lack of tools for Mccp genome engineering. In this work, key synthetic biology techniques recently developed for closely related mycoplasmas were adapted to Mccp. CReasPy-Cloning was used to simultaneously clone and engineer the Mccp genome in yeast, prior to whole-genome transplantation into M. capricolum subsp. capricolum recipient cells. This approach was used to knock out an S41 serine protease gene recently identified as a potential virulence factor, leading to the generation of the first site-specific Mccp mutants. The Cre-lox recombination system was then applied to remove all DNA sequences added during genome engineering. Finally, the resulting unmarked S41 serine protease mutants were validated by whole-genome sequencing and their non-caseinolytic phenotype was confirmed by casein digestion assay on milk agar. The synthetic biology tools that have been successfully implemented in Mccp allow the addition and removal of genes and other genetic features for the construction of seamless targeted mutants at ease, which will pave the way for both the identification of key pathogenicity determinants of Mccp and the rational design of novel, improved vaccines for the control of CCPP.

Multi-locus sequence analysis reveals great genetic diversity among Mycoplasma capricolum subsp. capripneumoniae strains in Asia.

Multi-Locus Sequence Analysis (MLSA) of Mycoplasma capricolum subsp. capripneumoniae (Mccp) strains from Asia revealed unforeseen diversity and a central position for genotyping groups representing strains from Central/East Asia, suggesting a possible origin of contagious caprine pleuropneumonia in this continent. A better assessment of the emergence, diversity and distribution of Mccp in Asia and Africa calls for renewed efforts to dramatically enlarge the sample of strains. Availability and affordability in the field, added to superior typeability (directly from poor samples) and high stability, discriminatory power and concordance with epidemiological and phylogenetic analyses, make MLSA an excellent tool for such investigations.

Proteases as Secreted Exoproteins in Mycoplasmas from Ruminant Lungs and Their Impact on Surface-Exposed Proteins.

Many mycoplasma species are isolated from the ruminant lungs as either saprophytes or true pathogens. These wall-less bacteria possess a minimal genome and reduced metabolic capabilities. Accordingly, they rely heavily on their hosts for the supply of essential metabolites and, notably, peptides. Seven of 13 ruminant lung-associated Mycoplasma (sub)species were shown to possess caseinolytic activity when grown in rich media and assessed with a quantitative fluorescence test. For some species, this activity was detected in spent medium, an indication that proteases were secreted outside the mycoplasma cells. To identify these proteases, we incubated concentrated washed cell pellets in a defined medium and analyzed the supernatants by tandem mass spectrometry. Secreted-protease activity was detected mostly in the species belonging to the Mycoplasma mycoides cluster (MMC) and, to a lesser extent, in Mycoplasma bovirhinis Analyzing a Mycoplasma mycoides subsp. capri strain, chosen as a model, we identified 35 expressed proteases among 55 predicted coding genes, of which 5 were preferentially found in the supernatant. Serine protease S41, acquired by horizontal gene transfer, was responsible for the caseinolytic activity, as demonstrated by zymography and mutant analysis. In an M. capricolum mutant, inactivation of the S41 protease resulted in marked modification of the expression or secretion of 17 predicted surface-exposed proteins. This is an indication that the S41 protease could have a role in posttranslational cleavage of surface-exposed proteins and ectodomain shedding, whose physiological impacts still need to be explored.IMPORTANCE Few studies pertaining to proteases in ruminant mycoplasmas have been reported. Here, we focus on proteases that are secreted outside the mycoplasma cell using a mass spectrometry approach. The most striking result is the identification, within the Mycoplasma mycoides cluster, of a serine protease that is exclusively detected outside the mycoplasma cells and is responsible for casein digestion. This protease may also be involved in the posttranslational processing of surface proteins, as suggested by analysis of mutants showing a marked reduction in the secretion of extracellular proteins. By analogy, this finding may help increase understanding of the mechanisms underlying this ectodomain shedding in other mycoplasma species. The gene encoding this protease is likely to have been acquired via horizontal gene transfer from Gram-positive bacteria and sortase-associated surface proteases. Whether this protease and the associated ectodomain shedding are related to virulence has yet to be ascertained.

Recommended rejection of the names Malacoplasma gen. nov., Mesomycoplasma gen. nov., Metamycoplasma gen. nov., Metamycoplasmataceae fam. nov., Mycoplasmoidaceae fam. nov., Mycoplasmoidales ord. nov., Mycoplasmoides gen. nov., Mycoplasmopsis gen. nov. [Gupta, Sawnani, Adeolu, Alnajar and Oren 2018] and all proposed species comb. nov. placed therein.

The consensus of the members of the International Committee on Systematics of Prokaryotes' Subcommittee on the taxonomy of Mollicutes is that recently proposed sweeping changes to nomenclature of members of the Mycoplasmatales, specifically involving introduction of the names Malacoplasma gen. nov., Mesomycoplasma gen. nov., Metamycoplasma gen. nov., Metamycoplasmataceaefam. nov., Mycoplasmoidaceaefam. nov., Mycoplasmoidalesord. nov., Mycoplasmoides gen. nov., Mycoplasmopsis gen. nov., and all proposed species or subspecies comb. nov. placed therein, should be rejected because they violate one or more essential points of the International Code of Nomenclature of Prokaryotes.

Highly dynamic genomic loci drive the synthesis of two types of capsular or secreted polysaccharides within the Mycoplasma mycoides cluster.

Mycoplasmas of the Mycoplasma mycoides cluster are all ruminant pathogens. Mycoplasma mycoides subsp. mycoides is responsible for contagious bovine pleuropneumonia and is known to produce capsular polysaccharide (CPS) and exopolysaccharide (EPS). Previous studies have strongly suggested a role for Mycoplasma mycoides subsp. mycoides polysaccharides in pathogenicity. Mycoplasma mycoides subsp. mycoides-secreted EPS was recently characterized as a ß(1→6)-galactofuranose homopolymer (galactan) identical to the capsular product. Here, we extended the characterization of secreted polysaccharides to all other members of the M. mycoides cluster: M. capricolum subsp. capripneumoniae, M. capricolum subsp. capricolum, M. leachii, and M. mycoides subsp. capri (including the LC and Capri serovars). Extracted EPS was characterized by nuclear magnetic resonance, resulting in the identification of a homopolymer of ß(1→2)-glucopyranose (glucan) in M. capricolum subsp. capripneumoniae and M. leachii. Monoclonal antibodies specific for this glucan and for the Mycoplasma mycoides subsp. mycoides-secreted galactan were used to detect the two polysaccharides. While M. mycoides subsp. capri strains of serovar LC produced only capsular galactan, no polysaccharide could be detected in strains of serovar Capri. All strains of M. capricolum subsp. capripneumoniae and M. leachii produced glucan CPS and EPS, whereas glucan production and localization varied among M. capricolum subsp. capricolum strains. Genes associated with polysaccharide synthesis and forming a biosynthetic pathway were predicted in all cluster members. These genes were organized in clusters within two loci representing genetic variability hot spots. Phylogenetic analysis showed that some of these genes, notably galE and glf, were acquired via horizontal gene transfer. These findings call for a reassessment of the specificity of the serological tests based on mycoplasma polysaccharides.

A large-scale genomic approach affords unprecedented resolution for the molecular epidemiology and evolutionary history of contagious caprine pleuropneumonia.

Contagious caprine pleuropneumonia (CCPP), caused by Mycoplasma capricolum subsp. capripneumoniae (Mccp), is a devastating disease of domestic goats and of some wild ungulate species. The disease is currently spreading in Africa and Asia and poses a serious threat to disease-free areas. A comprehensive view of the evolutionary history and dynamics of Mccp is essential to understand the epidemiology of CCPP. Yet, analysing the diversity of genetically monomorphic pathogens, such as Mccp, is complicated due to their low variability. In this study, the molecular epidemiology and evolution of CCPP was investigated using a large-scale genomic approach based on next-generation sequencing technologies, applied to a sample of strains representing the global distribution of this disease. A highly discriminatory multigene typing system was developed, allowing the differentiation of 24 haplotypes among 25 Mccp strains distributed in six genotyping groups, which showed some correlation with geographic origin. A Bayesian approach was used to infer the first robust phylogeny of the species and to date the principal events of its evolutionary history. The emergence of Mccp was estimated only at about 270 years ago, which explains the low genetic diversity of this species despite its high mutation rate, evaluated at 1.3 × 10(-6) substitutions per site per year. Finally, plausible scenarios were proposed to elucidate the evolution and dynamics of CCPP in Asia and Africa, though limited by the paucity of Mccp strains, particularly in Asia. This study shows how combining large-scale genomic data with spatial and temporal data makes it possible to obtain a comprehensive view of the epidemiology of CCPP, a precondition for the development of improved disease surveillance and control measures.

Free exopolysaccharide from Mycoplasma mycoides subsp. mycoides possesses anti-inflammatory properties.

In this study we explored the immunomodulatory properties of highly purified free galactan, the soluble exopolysaccharide secreted by Mycoplasma mycoides subsp. mycoides (Mmm). Galactan was shown to bind to TLR2 but not TLR4 using HEK293 reporter cells and to induce the production of the anti-inflammatory cytokine IL-10 in bovine macrophages, whereas low IL-12p40 and no TNF-α, both pro-inflammatory cytokines, were induced in these cells. In addition, pre-treatment of macrophages with galactan substantially reduced lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines TNF- and IL-12p40 while increasing LPS-induced secretion of immunosuppressive IL-10. Also, galactan did not activate naïve lymphocytes and induced only low production of the Th1 cytokine IFN-γ in Mmm-experienced lymphocytes. Finally, galactan triggered weak recall proliferation of CD4+ T lymphocytes from contagious bovine pleuropneumonia-infected animals despite having a positive effect on the expression of co-stimulatory molecules on macrophages. All together, these results suggest that galactan possesses anti-inflammatory properties and potentially provides Mmm with a mechanism to evade host innate and adaptive cell-mediated immune responses.

An international collaborative study to determine the prevalence of contagious caprine pleuropneumonia by monoclonal antibody-based cELISA.

BACKGROUND: Few serological tests are available for detecting antibodies against Mycoplasma capricolum subsp. capripneumoniae, the causal agent of contagious caprine pleuropneumonia (CCPP). The complement fixation test, the test prescribed for international trade purposes, uses a crude antigen that cross-reacts with all the other mycoplasma species of the "mycoides cluster" frequently infecting goat herds. The lack of a more specific test has been a real obstacle to the evaluation of the prevalence and economic impact of CCPP worldwide. A new competitive ELISA kit for CCPP, based on a previous blocking ELISA, was formatted at CIRAD and used to evaluate the prevalence of CCPP in some regions of Kenya, Ethiopia, Mauritius, Tajikistan and Pakistan in an international collaborative study. RESULTS: The strict specificity of the test was confirmed in CCPP-free goat herds exposed to other mycoplasma species of the "mycoides cluster". Prevalence studies were performed across the enzootic range of the disease in Africa and Asia. Seroprevalence was estimated at 14.6% in the Afar region of Ethiopia, whereas all the herds presented for CCPP vaccination in Kenya tested positive (individual seroprevalence varied from 6 to 90% within each herd). In Mauritius, where CCPP emerged in 2009, nine of 62 herds tested positive. In Central Asia, where the disease was confirmed only recently, no positive animals were detected in the Wakhan District of Afghanistan or across the border in neighboring areas of Tajikistan, whereas seroprevalence varied between 2.7% and 44.2% in the other districts investigated and in northern Pakistan. The test was also used to monitor seroconversion in vaccinated animals. CONCLUSIONS: This newly formatted CCPP cELISA kit has retained the high specificity of the original kit. It can therefore be used to evaluate the prevalence of CCPP in countries or regions without vaccination programs. It could also be used to monitor the efficacy of vaccination campaigns as high-quality vaccines induce high rates of seroconversion.

Interactions between Mycoplasma mycoides subsp. mycoides and bovine macrophages under physiological conditions.

We investigated the interactions of unopsonized and opsonized Mycoplasma mycoides subsp. mycoides (Mmm) with bovine macrophages in vitro. Mmm survived and proliferated extracellularly on bovine macrophage cell layers in the absence of Mmm-specific antisera. Bovine complement used at non-bactericidal concentrations did neither have opsonizing effect nor promoted intracellular survival, whereas Mmm-specific antisera substantially increased phagocytosis and Mmm killing. A phagocytosis-independent uptake of Mmm by macrophages occurred at a high multiplicity of infection, also found to induce the production of TNF, and both responses were unaffected by non-bactericidal doses of bovine complement. Bovine complement used at higher doses killed Mmm in cell-free cultures and completely abrogated TNF responses by macrophages. These results provide a framework to identify Mmm antigens involved in interactions with macrophages and targeted by potentially protective antibodies and point towards a pivotal role of complement in the control of inflammatory responses in contagious bovine pleuropneumonia.

Deep sequencing and variant frequency analysis for the quality control of a live bacterial vaccine against contagious bovine pleuropneumonia, strain T1.

Vaccination is the most cost-effective tool to control contagious bovine pleuropneumonia. The vaccines currently used in Africa are derived from a live strain called T1, which was attenuated by passage in embryonated eggs and broth culture. The number of passages is directly correlated to the degree of attenuation of the vaccinal strains and inversely correlated to their immunogenicity in cattle. Current quality control protocols applied to vaccine batches allow the assessment of identity, purity, and titers, but cannot assess the level of genetic drift form the parental vaccine strains. Deep sequencing was used to assess the genetic drift generated over controlled in vitro passages of the parental strain, as well as on commercial vaccine batches. Signatures of cloning procedures were detected in some batches, which imply a deviation from the standard production protocol. Deep sequencing is proposed as a new tool for the identity and stability control of T1 vaccines.

Combining Fusion of Cells with CRISPR-Cas9 Editing for the Cloning of Large DNA Fragments or Complete Bacterial Genomes in Yeast.

The genetic engineering of genome fragments larger than 100 kbp is challenging and requires both specific methods and cloning hosts. The yeast Saccharomyces cerevisiae is considered as a host of choice for cloning and engineering whole or partial genomes from viruses, bacteria, and algae. Several methods are now available to perform these manipulations, each with its own limitations. In order to extend the range of yeast cloning strategies, a new approach combining two already described methods, Fusion cloning and CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by the fusion of bacterial cells with yeast spheroplasts carrying the CRISPR-Cas9 system. With this new approach, we demonstrate the feasibility of cloning and editing whole genomes from several Mycoplasma species belonging to different phylogenetic groups. We also show that CReasPy-Fusion allows the capture of large genome fragments with high efficacy, resulting in the successful cloning of selected loci in yeast. We finally identify bacterial nuclease encoding genes as barriers for CReasPy-Fusion by showing that their removal from the donor genome improves the cloning efficacy.

Emergence of atypical Mycoplasma agalactiae strains harboring a new prophage and associated with an alpine wild ungulate mortality episode.

The bacterium Mycoplasma agalactiae is responsible for contagious agalactia (CA) in small domestic ruminants, a syndrome listed by the World Organization for Animal Health and responsible for severe damage to the dairy industry. Recently, we frequently isolated this pathogen from lung lesions of ibexes during a mortality episode in the French Alps. This situation was unusual in terms of host specificity and tissue tropism, raising the question of M. agalactiae emergence in wildlife. To address this issue, the ibex isolates were characterized using a combination of approaches that included antigenic profiles, molecular typing, optical mapping, and whole-genome sequencing. Genome analyses showed the presence of a new, large prophage containing 35 coding sequences (CDS) that was detected in most but not all ibex strains and has a homolog in Mycoplasma conjunctivae, a species causing keratoconjunctivitis in wild ungulates. This and the presence in all strains of large integrated conjugative elements suggested highly dynamic genomes. Nevertheless, M. agalactiae strains circulating in the ibex population were shown to be highly related, most likely originating from a single parental clone that has also spread to another wild ungulate species of the same geographical area, the chamois. These strains clearly differ from strains described in Europe so far, including those found nearby, before CA eradication a few years ago. While M. agalactiae pathogenicity in ibexes remains unclear, our data showed the emergence of atypical strains in Alpine wild ungulates, raising the question of a role for the wild fauna as a potential reservoir of pathogenic mycoplasmas.

Mycoplasma mycoides, from "mycoides Small Colony" to "capri". A microevolutionary perspective.

BACKGROUND: The Mycoplasma mycoides cluster consists of five species or subspecies that are ruminant pathogens. One subspecies, Mycoplasma mycoides subspecies mycoides Small Colony (MmmSC), is the causative agent of contagious bovine pleuropneumonia. Its very close relative, Mycoplasma mycoides subsp. capri (Mmc), is a more ubiquitous pathogen in small ruminants causing mastitis, arthritis, keratitis, pneumonia and septicaemia and is also found as saprophyte in the ear canal. To understand the genetics underlying these phenotypic differences, we compared the MmmSC PG1 type strain genome, which was already available, with the genome of an Mmc field strain (95010) that was sequenced in this study. We also compared the 95010 genome with the recently published genome of another Mmc strain (GM12) to evaluate Mmc strain diversity. RESULTS: The MmmSC PG1 genome is 1,212 kbp and that of Mmc 95010 is ca. 58 kbp shorter. Most of the sequences present in PG1 but not 95010 are highly repeated Insertion Sequences (three types of IS) and large duplicated DNA fragments. The 95010 genome contains five types of IS, present in fewer copies than in PG1, and two copies of an integrative conjugative element. These mobile genetic elements have played a key role in genome plasticity, leading to inversions of large DNA fragments. Comparison of the two genomes suggested a marked decay of the PG1 genome that seems to be correlated with a greater number of IS. The repertoire of gene families encoding surface proteins is smaller in PG1. Several genes involved in polysaccharide metabolism and protein degradation are also absent from, or degraded in, PG1. CONCLUSIONS: The genome of MmmSC PG1 is larger than that of Mmc 95010, its very close relative, but has less coding capacity. This is the result of large genetic rearrangements due to mobile elements that have also led to marked gene decay. This is consistent with a non-adaptative genomic complexity theory, allowing duplications or pseudogenes to be maintained in the absence of adaptive selection that would lead to purifying selection and genome streamlining over longer evolutionary times. These findings also suggest that MmmSC only recently adapted to its bovine host.

Fatal outbreak of Mycoplasma capricolum pneumonia in endangered markhors.

A pneumonia outbreak reduced the numbers of a wild population of endangered markhors (Capra falconeri) in Tajikistan in 2010. The infection was diagnosed by histologic examination and bacteriologic testing. Mycoplasma capricolum subsp. capricolum was the sole infectious agent detected. Cross-species transmission from domestic goats may have occurred.

Characterization of in vivo-acquired resistance to macrolides of Mycoplasma gallisepticum strains isolated from poultry.

The macrolide class of antibiotics, including tylosin and tilmicosin, is widely used in the veterinary field for prophylaxis and treatment of mycoplasmosis. In vitro susceptibility testing of 50 strains of M. gallisepticum isolated in Israel during the period 1997-2010 revealed that acquired resistance to tylosin as well as to tilmicosin was present in 50% of them. Moreover, 72% (13/18) of the strains isolated from clinical samples since 2006 showed acquired resistance to enrofloxacin, tylosin and tilmicosin. Molecular typing of the field isolates, performed by gene-target sequencing (GTS), detected 13 molecular types (I-XIII). Type II was the predominant type prior to 2006 whereas type X, first detected in 2008, is currently prevalent. All ten type X strains were resistant to both fluoroquinolones and macrolides, suggesting selective pressure leading to clonal dissemination of resistance. However, this was not a unique event since resistant strains with other GTS molecular types were also found. Concurrently, the molecular basis for macrolide resistance in M. gallisepticum was identified. Our results revealed a clear-cut correlation between single point mutations A2058G or A2059G in domain V of the gene encoding 23S rRNA (rrnA, MGA_01) and acquired macrolide resistance in M. gallisepticum. Indeed, all isolates with MIC ≥ 0.63 µg/mL to tylosin and with MIC ≥ 1.25 µg/mL to tilmicosin possess one of these mutations, suggesting an essential role in decreased susceptibility of M. gallisepticum to 16-membered macrolides.

Multi-locus sequence analysis of Mycoplasma capricolum subsp. capripneumoniae for the molecular epidemiology of contagious caprine pleuropneumonia.

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the causative agent of contagious caprine pleuropneumonia (CCPP), a devastating disease of domestic goats. The exact distribution of CCPP is not known but it is present in Africa and the Middle East and represents a significant threat to many disease-free areas including Europe. Furthermore, CCPP has been recently identified in Tajikistan and China. A typing method with an improved resolution based on Multi-Locus Sequence Analysis (MLSA) has been developed to trace new epidemics and to elucidate whether the recently identified cases in continental Asia were due to recent importation of Mccp. The H2 locus, a polymorphic region already in use as a molecular marker for Mccp evolution, was complemented with seven new loci selected according to the analysis of polymorphisms observed among the genome sequences of three Mccp strains. A total of 25 strains, including the two new strains from Asia, were analysed by MLSA resulting in the discrimination of 15 sequence types based on 53 polymorphic positions. A distance tree inferred from the concatenated sequences of the eight selected loci revealed two evolutionary lineages comprising five groups, which showed good correlation with geographic origins. The presence of a distinct Asian cluster strongly indicates that CCPP was not recently imported to continental Asia. It is more likely that the disease has been endemic in the area for a long time, as supported by historical clinical descriptions. In conclusion, this MLSA strategy constitutes a highly discriminative tool for the molecular epidemiology of CCPP.

A whole-genome worldwide molecular epidemiology approach for contagious caprine pleuropneumonia.

Contagious caprine pleuropneumonia is an infectious and contagious disease affecting goats and wildlife ruminants, mostly in Africa and Asia. It is caused by a mycoplasma, Mycoplasma capricolum susbp. capripneumoniae, which is very fastidious. This may be the reason why there are few reports of its isolation and characterization. This study describes the development of a whole genome typing strategy based on sequencing reads assemblies on a reference genome (Abomsa, GenBank accession LM995445) and extraction of informative single nucleotide polymorphism. FASTA sequences inferred from the variant calling files were used to establish a comprehensive phylogenetic tree based on 2880 SNPs. This tree included a total of 34 strains originating from all the regions where CCPP has been detected, as well as strains isolated from wildlife. A recent isolate from West-Niger was positioned closely to another 1995 East-Niger isolate, an indication that CCPP may be extending westward in Africa. Six 2013 Tanzanian isolates had identical sequences in spite of diverse geographical origins. This could be explained by the clonal expansion of a virulent strain at that time in East Africa. Although all strains isolated from wildlife in the Middle East were in the same phylogenetic group, this may not sign an adaptation to new hosts. The most probable explanation for wildlife contamination remains the contact with goats. This strategy will easily accommodate new data in the near future and should become a gold-standard high-resolution typing procedure for the surveillance of contagious caprine pleuropneumonia.

Mycoplasmas are no exception to extracellular vesicles release: Revisiting old concepts.

Release of extracellular vesicles (EV) by Gram-negative and positive bacteria is being frequently reported. EV are nano-sized, membrane-derived, non-self-replicating, spherical structures shed into the extracellular environment that could play a role in bacteria-host interactions. Evidence of EV production in bacteria belonging to the class Mollicutes, which are wall-less, is mainly restricted to the genus Acholeplasma and is scanty for the Mycoplasma genus that comprises major human and animal pathogens. Here EV release by six Mycoplasma (sub)species of clinical importance was investigated. EV were obtained under nutritional stress conditions, purified by ultracentrifugation and observed by electron microscopy. The membrane proteins of EV from three different species were further identified by mass spectrometry as a preliminary approach to determining their potential role in host-pathogen interactions. EV were shown to be released by all six (sub)species although their quantities and sizes (30-220 nm) were very variable. EV purification was complicated by the minute size of viable mycoplasmal cells. The proteins of EV-membranes from three (sub)species included major components of host-pathogen interactions, suggesting that EV could contribute to make the host-pathogen interplay more complex. The process behind EV release has yet to be deciphered, although several observations demonstrated their active release from the plasma membrane of living cells. This work shed new light on old concepts of "elementary bodies" and "not-cell bound antigens".

Unexpected field observations and transmission dynamics of contagious caprine pleuropneumonia in a sand gazelle herd.

Contagious caprine pleuropneumonia (CCPP), caused by Mycoplasma capricolum subsp. capripneumoniae, has long been considered a goat-specific disease. Since 2007 there has been growing evidence that this disease can affect wild ungulates either kept in captivity or in the wild. In 2013, a large collection of sand gazelles (Gazella marica) held in the United Arab Emirates suffered heavy losses due to a CCPP epizootic confirmed by PCR and isolation. Animals displayed typical lesions, with unilateral pneumonia and profuse pleurisy. An initial antibiotic treatment consisting of tylosin administered in drinking water did not improve the animals' condition and vaccination failed to stop the spread to contiguous pens. A treatment with tetracycline mixed in feed pellets finally succeeded to stop the evolution of the disease. A subsequent vaccine trial, performed on naïve animals, showed that only a reference CCPP vaccine produced according to OIE standards induced a sero-conversion by CCPP competition ELISA, while the commercially available vaccines did not. A SEIRD compartment transmission model was developed to better understand the dynamics of the disease. The parameters were initially set as per expert opinion and then adjusted to fit the observed mortality data. The basic reproductive number R0 was estimated to be between 2.3-2.7, while the final mortality rate reached up to 70% in some pens. Transmission of infectious droplets from an external source, through a distance of at least the 50 m separating the pens from the perimeter fence, remains the most plausible explanation for the contamination of this stock of gazelles.

Selective medium for culture of Mycoplasma hyopneumoniae.

The fastidious porcine respiratory pathogen Mycoplasma hyopneumoniae has proven difficult to culture since it was first isolated in 1965. A reliable solid medium has been particularly challenging. Moreover, clinical and pathological samples often contain the fast-growing M. hyorhinis which contaminates and overgrows M. hyopneumoniae in primary culture. The aim of this study was to optimise the culture medium for recovery of M. hyopneumoniae and to devise a medium for selection of M. hyopneumoniae from clinical samples also containing M. hyorhinis. The solid medium devised by Niels Friis was improved by use of Purified agar and incorporation of DEAE-dextran. Addition of glucose or neutralization of acidity in liquid medium with NaOH did not improve the final yield of viable organisms or alter the timing of peak viability. Analysis of the relative susceptibility of M. hyopneumoniae and M. hyorhinis strains to four antimicrobials showed that M. hyopneumoniae is less susceptible than M. hyorhinis to kanamycin. This was consistent in all UK and Danish strains tested. A concentration of 2µg/ml of kanamycin selectively inhibited the growth of all M. hyorhinis tested, while M. hyopneumoniae was able to grow. This forms the basis of an effective selective culture medium for M. hyopneumoniae.