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During the evolution of SARS-CoV-2 in humans, a D614G substitution in the spike glycoprotein (S) has emerged; virus containing this substitution has become the predominant circulating variant in the COVID-19 pandemic1. However, whether the increasing prevalence of this variant reflects a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains unknown. Here we use isogenic SARS-CoV-2 variants to demonstrate that the variant that contains S(D614G) has enhanced binding to the human cell-surface receptor angiotensin-converting enzyme 2 (ACE2), increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a human ACE2 knock-in mouse model, and markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Our data show that the D614G substitution in S results in subtle increases in binding and replication in vitro, and provides a real competitive advantage in vivo—particularly during the transmission bottleneck. Our data therefore provide an explanation for the global predominance of the variant that contains S(D614G) among the SARS-CoV-2 viruses that are currently circulating. A SARS-CoV-2 variant containing a D614G substitution in the spike protein shows enhanced binding to human ACE2, increased replication in human cell cultures and a competitive advantage in animal models of infection.

SARS-CoV-2 is a highly contagious respiratory virus and the causative agent for COVID-19. The severity of disease varies from mildly symptomatic to lethal and shows an extraordinary correlation with increasing age, which represents the major risk factor for severe COVID-191. However, the precise pathomechanisms leading to aggravated disease in the elderly are currently unknown. Delayed and insufficient antiviral immune responses early after infection as well as dysregulated and overshooting immunopathological processes late during disease were suggested as possible mechanisms. Here we show that the age-dependent increase of COVID-19 severity is caused by the disruption of a timely and well-coordinated innate and adaptive immune response due to impaired interferon (IFN) responses. To overcome the limitations of mechanistic studies in humans, we generated a mouse model for severe COVID-19 and compared the kinetics of the immune responses in adult and aged mice at different time points after infection. Aggravated disease in aged mice was characterized by a diminished IFN-γ response and excessive virus replication. Accordingly, adult IFN-γ receptor-deficient mice phenocopied the age-related disease severity and supplementation of IFN-γ reversed the increased disease susceptibility of aged mice.Mimicking impaired type I IFN immunity in adult and aged mice, a second major risk factor for severe COVID-192–4, we found that therapeutic treatment with IFN-λ in adult and a combinatorial treatment with IFN-γ and IFN-λ in aged Ifnar1-/-mice was highly efficient in protecting against severe disease.Our findings provide an explanation for the age-dependent disease severity of COVID-19 and clarify the nonredundant antiviral functions of type I, II and III IFNs during SARS-CoV-2 infection in an age-dependent manner. Based on our data, we suggest that highly vulnerable individuals combining both risk factors, advanced age and an impaired type I IFN immunity, may greatly benefit from immunotherapy combining IFN-γ and IFN-λ.

Digital dermatitis (DD) is a major infectious foot disease of cattle worldwide. Some DD stages are associated with lameness, and the disease has significant economic and animal welfare consequences. The pathogenesis of the disease is not yet fully understood, but Treponema spp. have been associated consistently with clinical cases. Isolation of these fastidious bacteria is difficult and cumbersome. We describe an improved method enabling the culturing of the 3 Treponema spp. (T. pedis, T. phagedenis, and T. medium) from bovine foot specimens derived from DD lesions, using a combination of membrane filtering and subsequent growth on selective agar media. The entire procedure from sampling to verification of individual Treponema spp. takes up to 24 d. In addition, we established a MALDI-TOF MS–based identification method to be applied for confirmation of the different Treponema spp. This scheme provides an unambiguous, simple, and straightforward identification procedure for DD-associated Treponema spp.

Respiratory diseases negatively impact the global goat industry, but are understudied. There is a shortage of established and biological relevant in vitro or ex vivo assays to study caprine respiratory infections. Here, we describe the establishment of an in vitro system based on well-differentiated caprine airway epithelial cell (AEC) cultures grown under air liquid interface conditions as an experimental platform to study caprine respiratory pathogens. The functional differentiation of the AEC cultures was monitored and confirmed by light and immunofluorescence microscopy, scanning electron microscopy and examination of histological sections. We validated the functionality of the platform by studying Influenza D Virus (IDV) infection and Mycoplasma mycoides subsp. capri (Mmc) colonization over 5 days, including monitoring of infectious agents by titration and qPCR as well as colour changing units, respectively. The inoculation of caprine AEC cultures with IDV showed that efficient viral replication takes place, and revealed that IDV has a marked cell tropism for ciliated cells. Furthermore, AEC cultures were successfully infected with Mmc using a multiplicity of infection of 0.1 and colonization was monitored over several days. Altogether, these results demonstrate that our newly-established caprine AEC cultures can be used to investigate host-pathogen interactions of caprine respiratory pathogens.

Safe and effective vaccines are urgently needed to stop the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We construct a series of live attenuated vaccine candidates by large-scale recoding of the SARS-CoV-2 genome and assess their safety and efficacy in Syrian hamsters. Animals were vaccinated with a single dose of the respective recoded virus and challenged 21 days later. Two of the tested viruses do not cause clinical symptoms but are highly immunogenic and induce strong protective immunity. Attenuated viruses replicate efficiently in the upper but not in the lower airways, causing only mild pulmonary histopathology. After challenge, hamsters develop no signs of disease and rapidly clear challenge virus: at no time could infectious virus be recovered from the lungs of infected animals. The ease with which attenuated virus candidates can be produced and administered favors their further development as vaccines to combat the ongoing pandemic., Graphical abstract, Trimpert et al. construct safe and efficacious live attenuated SARS-CoV-2 vaccine candidates by recoding the SARS-CoV-2 genome. The lead vaccine candidate, sCPD9, is apathogenic in Syrian and Roborovski dwarf hamsters. A single intranasal droplet vaccination with sCPD9 induces strong neutralizing antibody responses and protects hamsters from disease caused by SARS-CoV-2.

Mycoplasma ovipneumoniae infects both sheep and goats causing pneumonia resulting in considerable economic losses worldwide. Current diagnosis methods such as bacteriological culture, serology, and PCR are time consuming and require sophisticated laboratory setups. Here we report the development of two rapid, specific and sensitive assays; an isothermal DNA amplification using recombinase polymerase amplification (RPA) and a real-time PCR for the detection of M. ovipneumoniae. The target for both assays is a specific region of gene WP_069098309.1, which encodes a hypothetical protein and is conserved in the genome sequences of ten publicly available M. ovipneumoniae strains. The RPA assay performed well at 39°C for 20 min and was combined with a lateral flow dipstick (RPA-LFD) for easy visualization of the amplicons. The detection limit of the RPA-LFD assay was nine genome copies of M. ovipneumoniae per reaction and was comparable to sensitivity of the real-time PCR assay. Both assays showed no cross-reaction with 38 other ovine and caprine pathogenic microorganisms and two parasites of ruminants, demonstrating a high degree of specificity. The assays were validated using bronchoalveolar lavage fluid and nasal swab samples collected from sheep. The positive rate of RPA-LFD (97.4%) was higher than the real-time PCR (95.8%) with DNA as a template purified from the clinical samples. The RPA assay was significantly better at detecting M. ovipneumoniae in clinical samples compared to the real-time PCR when DNA extraction was omitted (50% and 34.4% positive rate for RPA-LFD and real-time PCR respectively). The RPA-LFD developed here allows easy and rapid detection of M. ovipneumoniae infection without DNA extraction, suggesting its potential as a point-of-care test for field settings.

Summary This protocol describes a synthetic genomics pipeline to clone and engineer the entire 190-kbp genome of the African swine fever virus (ASFV) genotype II in yeast using transformation-associated recombination cloning. The viral genome was cloned using DNA directly extracted from a clinical sample. In addition, the precise deletion of a non-essential gene and its replacement by a synthetic reporter gene cassette are presented. This protocol is applicable to other ASFV genotypes and other large DNA viruses., Graphical abstract, Highlights • Use of TAR for the individual cloning of five ASFV sub-genomic fragments in yeast • Chemical synthesis of both 5′ and 3′ ITR genomic regions • Replacement of the C962R gene by reporter genes (eGFP or secNLuc), This protocol describes a synthetic genomics pipeline to clone and engineer the entire 190-kbp genome of the African swine fever virus (ASFV) genotype II in yeast using transformation-associated recombination cloning. The viral genome was cloned using DNA directly extracted from a clinical sample. In addition, the precise deletion of a non-essential gene and its replacement by a synthetic reporter gene cassette are presented. This protocol is applicable to other ASFV genotypes and other large DNA viruses.

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.

Postmortem examination and immunohistochemical and bacteriologic analyses on a free-ranging European red squirrel (Sciurus vulgaris) revealed a systemic infection with Francisella tularensis. Genome sequencing and single-nucleotide polymorphism (SNP) analysis were consistent with F. tularensis subs. holarctica clade B.45. Tularemia has not previously been reported in this species.

Francisella tularensis subsp. holarctica is a select agent causing life-threatening tularemia. It has been isolated from humans and animals, mainly lagomorphs and rodents, rarely other wild carnivore species. Increasing numbers of human tularemia cases have been reported during the last 5 years in Switzerland. Here we report the first isolation of Francisella tularensis subsp. holarctica from a domestic cat in Europe and compare its genome sequence with other Swiss isolates. The cat isolate shows a close phylogenetic relationship with a contemporary hare isolate from close geographic proximity, indicating a possible epidemiological link.

Highlights • A panel of anti-Mmm mAbs was produced and screened for host-pathogen inhibition. • 13 mAbs inhibited adhesion of Mmm to host target cells. • Anti-capsular polysaccharide inhibited growth and caused agglutination of Mmm. • Anti-PDHC inhibited adherence of Mmm cells showing the possible role of glycolytic enzymes in host-pathogen interaction. • One novel antigen that is a promising vaccine candidate against CBPP identified., Mycoplasma mycoides subspecies mycoides (Mmm) adhesion is tissue and host specific. Inhibition of adhesion will prevent Mmm from binding to lung cells and hence prevent colonization and disease. The aim of this study was to develop a panel of Mmm monoclonal antibodies against Mmm and use these antibodies to investigate their inhibitory effect on the adherence of Mmm to bovine lung epithelial cells (BoLEC), and to further identify an antigen to any of the inhibitory antibodies. Thirteen anti-Mycoplasma mycoides subsp. mycoides (AMMY) monoclonal antibodies (mAbs) inhibited adhesion by at least 30% and ten of the mAbs bound to multiple bands on Western blots suggesting that the antibodies bound to proteins of variable sizes. AMMY 10, a previously characterized Mmm- capsular polysaccharide (CPS) specific antibody, inhibited growth of Mmm in vitro and also caused agglutination of Mmm total cell lysate. AMMY 5, a 2-oxo acid dehydrogenase acyltransferase (Catalytic domain) (MSC_0267) specific antibody, was identified and polyclonal rabbit serum against recombinant MSC_0267 blocked adhesion of Mmm to BoLEC by 41%. Antigens recognized by these antibodies could be vaccine candidate(s) and should be subsequently tested for their ability to induce a protective immune response in vivo.

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., 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.

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.

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.

Many Mycoplasma species have been shown to possess capsular polysaccharides (CPSs). We challenged goats with a CPS-defective or its CPS-possessing parental Mycoplasma mycoides strain. The CPS-defective strain was attenuated, demonstrating that the CPS consisting of galactofuranose is a virulence trait., 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.

In East Africa, contagious caprine pleuropneumonia (CCPP) is rated by farmers as one of the top priority diseases in livestock, higher than contagious bovine pleuropneumonia (CBPP). In July 2019, several stakeholders held a workshop at the International Livestock Research Institute’s Kapiti Ranch in Kenya to obtain feedback on the use of the current CCPP bacterin vaccine, with the aim to gain a better understanding of whether a genetically modified live CCPP vaccine could be registered for use and its possible implementation. …

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-1β, 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-1β, 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. Electronic supplementary material The online version of this article (doi:10.1007/s11250-016-0994-9) contains supplementary material, which is available to authorized users.

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

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.

The hepatitis C virus (HCV) is a major human pathogen. Genetically related viruses in animals suggest a zoonotic origin of HCV. The closest relative of HCV is found in horses (termed equine hepacivirus [EqHV]). However, low EqHV genetic diversity implies relatively recent acquisition of EqHV by horses, making a derivation of HCV from EqHV unlikely. To unravel the EqHV evolutionary history within equid sister species, we analyzed 829 donkeys and 53 mules sampled in nine European, Asian, African, and American countries by molecular and serologic tools for EqHV infection. Antibodies were found in 278 animals (31.5%), and viral RNA was found in 3 animals (0.3%), all of which were simultaneously seropositive. A low RNA prevalence in spite of high seroprevalence suggests a predominance of acute infection, a possible difference from the mostly chronic hepacivirus infection pattern seen in horses and humans. Limitation of transmission due to short courses of infection may explain the existence of entirely seronegative groups of animals. Donkey and horse EqHV strains were paraphyletic and 97.5 to 98.2% identical in their translated polyprotein sequences, making virus/host cospeciation unlikely. Evolutionary reconstructions supported host switches of EqHV between horses and donkeys without the involvement of adaptive evolution. Global admixture of donkey and horse hepaciviruses was compatible with anthropogenic alterations of EqHV ecology. In summary, our findings do not support EqHV as the origin of the significantly more diversified HCV. Identification of a host system with predominantly acute hepacivirus infection may enable new insights into the chronic infection pattern associated with HCV. IMPORTANCE The evolutionary origins of the human hepatitis C virus (HCV) are unclear. The closest animal-associated relative of HCV occurs in horses (equine hepacivirus [EqHV]). The low EqHV genetic diversity implies a relatively recent acquisition of EqHV by horses, limiting the time span for potential horse-to-human infections in the past. Horses are genetically related to donkeys, and EqHV may have cospeciated with these host species. Here, we investigated a large panel of donkeys from various countries using serologic and molecular tools. We found EqHV to be globally widespread in donkeys and identify potential differences in EqHV infection patterns, with donkeys potentially showing enhanced EqHV clearance compared to horses. We provide strong evidence against EqHV cospeciation and for its capability to switch hosts among equines. Differential hepacivirus infection patterns in horses and donkeys may enable new insights into the chronic infection pattern associated with HCV.

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.

Contagious Bovine Pleuropneumonia caused by Mycoplasma mycoides subsp. mycoides small colony type is a respiratory disease of considerable economic importance in sub-Saharan Africa; control of the disease in Africa is hampered by diagnostic tests which are suited for herd-level but not for individual animal diagnostics. In the work presented we identified 22 potential immunogenic antigens of the Kenyan outbreak strain B237 by using phage display technology. We determined the relative strength of immunogenicity, the discriminatory capacity between bovine positive and negative sera, and the cross-reactivity with rabbit hyperimmune sera directed against 15 different mycoplasmal species. The three best-performing antigens, a conserved hypothetical protein (MSC_0636), a glycosyl transferase (MSC_0108), and an acyl carrier protein phosphodiesterase (MSC_0029) were considered candidate diagnostic proteins. They were expressed as GST-fusion proteins in Escherichia coli, purified, and used in an ELISA as solid phase antigens. The diagnostic potential of the recombinant antigens was tested using the sera of ten experimentally infected animals and six control animals. This prototype test resulted in 100% diagnostic sensitivity and specificity. In comparison, the complement fixation test and the competitive ELISA performed with a diagnostic sensitivity of 70% and 60%, respectively.

Contagious bovine pleuropneumonia (CBPP) is a lung disease caused by the bacterial pathogen Mycoplasma mycoides ssp. mycoides small colony type (MmmSC). It has been spreading due to a number of factors including poor vaccine efficacy and poor sensitivity of current diagnostic tests. The purpose of this study was to assess interferon gamma (IFN-gamma) release after stimulation of peripheral blood mononuclear cells (PBMC) from experimentally infected cattle. PBMC collected from 15 artificially infected animals were incubated with different concentrations of total MmmSC antigen. After 72h of incubation the IFN-gamma release was measured and found to be elevated in 11 animals. We did not observe a correlation between IFN-gamma release of animals with and without pathomorphological gross lesions. Therefore, our data do not confirm a role for CD4 T-lymphocytes in protection, since there is no correlation between IFN-g secretion (supposed to be mainly derived from CD4 T-cells) and disease severity. Additionally, we applied immunocytochemistry on affected lung tissue and detected no build up of T-lymphocytes (CD4 T-cells, CD8 T-cells) but a high presence of myeloid cells.

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 (

International audience; 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 10(8) 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.

International audience; Mycoplasma mycoides subsp. capri (Mmc) and subsp. mycoides (Mmm) are important ruminant pathogens worldwide causing diseases such as pleuropneumonia, mastitis and septicaemia. They express galactofuranose residues on their surface, but their role in pathogenesis has not yet been determined. The M. mycoides genomes contain up to several copies of the glf gene, which encodes an enzyme catalyzing the last step in the synthesis of galactofuranose. We generated a deletion of the glf gene in a strain of Mmc using genome transplantation and tandem repeat endonuclease coupled cleavage (TREC) with yeast as an intermediary host for the genome editing. As expected, the resulting YCp1.1-Δglf strain did not produce the galactofuranose containing glycans as shown by immunoblots and immuno-electronmicroscopy employing a galactofuranose specific monoclonal antibody. The mutant lacking galactofuranose exhibited a decreased growth rate and a significantly enhanced adhesion to small ruminant cells. The mutant was also "leaking" as revealed by a β-galactosidase-based assay employing a membrane impermeable substrate. These findings indicate that galactofuranose-containing polysaccharides conceal adhesins and are important for membrane integrity. Unexpectedly, the mutant strain showed increased serum resistance.

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 Afade. Two of the groups immunized with five proteins each showed protection after the Mmm challenge (Groups A and C; P

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 naive 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.

Infections with verotoxin-producing Escherichia coli (VTEC) has resulted in increasing numbers of human illnesses annually. These illnesses usually result from the ability of VTEC to cause the attaching and effacing lesions (AE lesion). The AE phenotype is encoded by the locus of enterocyte effacement (LEE) pathogenicity island. A key adhesion factor involved is the outer membrane protein intimin, encoded by the eae gene within the LEE. Intimin types alpha, beta, gamma, delta, and epsilon have been described previously. Each intimin represents distinct phylogenetic lineages of LEE-positive strains. A new intimin type zeta was identified in a VTEC strain of the serotype O84:NM (nonmotile) that was isolated from a calf with diarrhea. zeta intimin showed the highest similarity (88%) of its amino acid sequence to the alpha intimin. For diagnostic purposes, we established a polymerase chain reaction (PCR) method for diagnosis of the key virulence traits of VTEC (i.e., verotoxins and intimins). This method also distinguishes between the toxins (VT1 and VT2) and the six intimin types. By applying the PCR method, intimin zeta in strains of other VTEC serotypes O84:H2, O92:NM, O119:H25, and O150:NM was identified. Because the intimin types represent distinctive phylogenetic E. coli lineages, application of the intimin subtyping PCR offers significant benefits. These include improving diagnosis of VTEC infection and increasing the understanding of evolution of attaching and effacing VTEC and other LEE-positive bacteria.

We have recently shown that the locus of enterocyte effacement (LEE) of the bovine enterohemorrhagic E. coli RW1374 (O103 : H2) resides within a large pathogenicity island (PAI), integrated in the vicinity of the phenylalanine tRNA gene pheV . Here we describe an additional, but LEE-negative genomic island in RW1374 in the vicinity of another phenylalanine tRNA gene, pheU , the sequence of which is identical to pheV . These two genomic islands revealed identity of the left, but a relative variability of their right end sequences. To investigate the mechanism of LEE-PAI distribution in E. coli , we analysed similar junctions in the pheU / pheV loci of additional EPEC and EHEC strains the LEE location of which had not been determined before. By hybridisation of NotI restriction fragments with probes specific for LEE, pheV locus, and pheU locus, the LEE was found linked to either one of these two loci. The results agreed well with recently published phylogenetic data and indicate that in the clones of diarrheagenic E. coli (Dec) Dec 11 and Dec 12, forming the phylogenetic cluster EPEC 2, and in the strains of the most typical serotypes of the Dec 8, belonging to the phylogenetic cluster EHEC 2, the LEE was linked with pheV and not with the pheU locus as previously assumed. Sequence comparison with other pheU - and pheV -located genomic islands from different E. coli pathotypes (uropathogenic E. coli , septicemic E. coli ) as well as from Shigella indicated the same structural features at the junctions. These conserved structures suggested a common DNA cassette, serving as common vehicle for horizontal gene transfer of various PAIs. In addition, the elements suggest an origin from a common pheU -located ancestor and integration into the chromosome through site-specific recombination. Our results indicate that pheU/pheV -located genomic islands played an important role in the evolution of several PAIs in E. coli and related pathogens.

Contagious bovine pleuropneumonia (CBPP) is a cattle disease that has hampered the development of the livestock sector in sub-Saharan Africa. Currently, vaccination with a live vaccine strain is its recommended control measure although unofficial antimicrobial use is widely practiced. Here, modelling techniques are used to assess the potential impact of early elimination of infected cattle via accurate diagnosis on CBPP dynamics. A herd-level stochastic epidemiological model explicitly incorporating test sensitivity and specificity is developed. Interventions by annual vaccination, annual testing and elimination and a combination of both are implemented in a stepwise manner and their effectiveness compared by running 1000 simulations per intervention over ten years. The model predicts that among the simulated interventions, the ones likely to eliminate the disease from an isolated herd all involved annual vaccination of more than 75% of the animals with a vaccine that protects for at least 18 months combined with annual testing (and elimination of positive reactors) of 75% of the animals every six months after vaccination. The highest probability of disease elimination was 97.5% and this could occur within a median of 2.3 years. Generally, our model predicts that regular testing and elimination of positive reactors using improved tests will play a significant role in minimizing CBPP burden especially in the current situation where improved vaccines are yet to be developed.

Background Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a respiratory disease of cattle, whereas the closely related Mycoplasma mycoides subsp. capri (Mmc) is a goat pathogen. Cyto-adherence is a crucial step in host colonization by mycoplasmas and subsequent pathogenesis. The aim of this study was to investigate the interactions between Mmm and mammalian host cells by establishing a cyto-adherence flow cytometric assay and comparing tissue and species specificity of Mmm and Mmc strains. Results There were little significant differences in the adherence patterns of eight different Mmm strains to adult bovine lung epithelial cells. However, there was statistically significant variation in binding to different host cells types. Highest binding was observed with lung epithelial cells, intermediate binding with endothelial cells and very low binding with fibroblasts, suggesting the presence of effective adherence of Mmm on cells lining the airways of the lung, which is the target organ for this pathogen, possibly by high expression of a specific receptor. However, binding to bovine fetal lung epithelial cells was comparably low; suggesting that the lack of severe pulmonary disease seen in many infected young calves can be explained by reduced expression of a specific receptor. Conclusions Mmm bound with high efficiency to adult bovine lung cells and less efficiently to calves or goat lung cells. The data show that cyto-adherence of Mmm is species- and tissue- specific confirming its role in colonization of the target host and subsequent infection and development of CBPP. Electronic supplementary material The online version of this article (doi:10.1186/s12917-015-0347-3) contains supplementary material, which is available to authorized users.

Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides (Mmm) is an economically very important cattle disease in sub-Saharan Africa. CBPP impacts animal health and poverty of livestock-dependent people through decreased animal productivity, reduced food supply, and the cost of control measures. CBPP is a barrier to trade in many African countries and this reduces the value of livestock and the income of many value chain stakeholders. The presence of CBPP also poses a constant threat to CBPP-free countries and creates costs in terms of the measures necessary to ensure the exclusion of disease. This opinion focuses on the biomedical research needed to foster the development of better control measures for CBPP. We suggest that different vaccine development approaches are followed in parallel. Basic immunology studies and systematic OMICs studies will be necessary in order to identify the protective arms of immunity and to shed more light on the pathogenicity mechanisms in CBPP. Moreover a robust challenge model and a close collaboration with African research units will be crucial to foster and implement a new vaccine for the progressive control of this cattle plague.

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.

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.

Background Contagious Bovine Pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides, is widespread in sub-Saharan Africa. The current live vaccine T1/44 has limited efficacy and occasionally leads to severe side effects in the animals. A better understanding of the immune responses triggered by Mycoplasma mycoides subsp. mycoides and their role in disease progression will help to facilitate the design of a rational vaccine. Currently, knowledge of cytokines involved in immunity and immunopathology in CBPP is rather limited. The aim of this study was to characterize the in vivo plasma concentrations of the cytokines TNF-α, IFN-γ, IL-4, IL-10 and the overall role of CD4+ T cells in the development of cytokine levels during a primary infection. Plasma cytokine concentrations in two groups of cattle (CD4+ T cell-depleted and non-depleted cattle) experimentally infected with Mycoplasma mycoides subsp. mycoides were measured and their relationship to the clinical outcomes was investigated. Results Plasma cytokine concentrations varied between animals in each group. Depletion of CD4+ T cells did not induce significant changes in plasma levels of TNF-α, IL-4, and IL-10, suggesting a minor role of CD4+ T cells in regulation or production of the three cytokines during the time window of depletion (1-2 weeks post depletion). Unexpectedly, the IFN-γ concentrations were slightly, but statistically significantly higher in the depleted group (p < 0.05) between week three and four post infection. Three CD4+ T cell-depleted animals that experienced severe disease, had high levels of TNF-α and IFN-γ. Only one severely diseased non-depleted animal showed a high serum concentration of IL-4 post infection. Conclusions Comparison of most severely diseased animals, which had to be euthanized prior to the expected date, versus less severe diseased animals, irrespective of the depletion status, suggested that high TNF-α levels are correlated with more severe pathology in concomitance with high IFN-γ levels.

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

Background Mycoplasma mycoides subsp. mycoides SC is the pathogenic agent of contagious bovine pleuropneumonia (CBPP), the most important disease of cattle in Africa causing significant economic losses. The re-emergence of CBPP in Europe in the 1980s and 1990s illustrates that it is still a threat also to countries that have successfully eradicated the disease in the past. Nowadays, probe-based real-time PCR techniques are among the most advanced tools for a reliable identification and a sensitive detection of many pathogens, but only few protocols have been published so far for CBPP diagnosis. Therefore we developed a novel TaqMan®-based real-time PCR assay comprising the amplification of two independent targets (MSC_0136 and MSC_1046) and an internal exogenous amplification control in a multiplex reaction and evaluated its diagnostic performance with clinical samples. Results The assays detected 49 MmmSC strains from diverse temporal and geographical origin, but did not amplify DNA from 82 isolates of 20 non-target species confirming a specificity of 100%. The detection limit was determined to be 10 fg DNA per reaction for the MSC_0136 assay and 100 fg per reaction for the MSC_1046 assay corresponding to 8 and 80 genome equivalents, respectively. The diagnostic performance of the assay was evaluated with clinical samples from 19 experimentally infected cattle and from 20 cattle without CBPP and compared to those of cultivation and a conventional PCR protocol. The two rt-PCR tests proved to be the most sensitive methods and identified all 19 infected animals. The different sample types used were not equally suitable for MmmSC detection. While 94.7% of lung samples from the infected cohort were positively tested in the MSC_0136 assay, only 81% of pulmonal lymph nodes, 31% of mediastinal lymph nodes and 25% of pleural fluid samples gave a positive result. Conclusions The developed multiplex rt-PCR assay is recommended as an efficient tool for rapid confirmation of a presumptive CBPP diagnosis in a well-equipped laboratory environment.

Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) has been eradicated in the developed world, but it is still present in many countries of sub-Saharan Africa. After initially successful control measures in the 1960s it has been spreading due to a lack of money, fragmentation of veterinary services, uncontrolled cattle movement, insufficient vaccine efficacy and sensitivity of current diagnostic tests. In this study we used two-dimensional polyacrylamide gel electrophoresis followed by immunoblot with sera from MmmSC-infected animals and MALDI-ToF mass spectrometry to identify novel immunogenic proteins as candidate molecules for improved diagnostics and vaccines. We identified 24 immunogens recognized by pooled sera from experimentally infected cattle. Furthermore, a serum from an animal with acute clinical disease as well as severe pathomorphological lesions recognized 13 additional immunogens indicating variation in the antibody responses to CBPP amongst cattle. Most immunogens showed compelling similarity to protein/gene sequences in the two ruminant pathogens M. capricolum subsp. capricolum and M. mycoides subsp. mycoides large colony type both belonging to the mycoides cluster. Three of these proteins, namely glycerol-3-phosphate oxidase, adenylosuccinate synthase, and glyceraldehyde-3-phosphate dehydrogenase, had no compelling homologue in the other distantly related bovine pathogen M. agalactiae. In addition, translation elongation factor Tu, heat shock protein 70, pyruvate dehydrogenase, and FKBP-type peptidyl-prolyl isomerase, which have been found to mediate adhesion to host tissue in other mycoplasmas were shown to be expressed and recognized by sera. These proteins have potential for the development of improved diagnostic tests and possibly vaccines.

To obtain insight into the occurrence and prevalence of Clostridium perfringens and its major toxins in polar bears (Ursus maritimus), we took fecal samples for bacteriologic analysis from live-captured bears in the Svalbard Archipelago, Norway, in 2001. Clostridium perfringens was isolated from 40 of 92 samples (44%). Thirty strains were further characterized by determining toxin type and were classified to be type A, while one was also positive for the gene encoding beta2-toxin. Despite the fact that C. perfringens type A has been associated with fatal diseases in several animal species as well as in humans, our data indicate that C. perfringens type A is an normal inhabitant of the gastrointestinal tract of polar bears.

Human infections with enterohemorrhagic E. coli (EHEC) strains of serotype O103:H2 are of increasing importance in Germany. As bovines are the principal EHEC reservoir behind the occurrence of human infections, we analyzed a pathogenicity island (PAI I(RW1374)) of bovine O103:H2 strain RW1374 to identify putative virulence features. This PAI I(RW1374) harbors a functional 34-kb locus of enterocyte effacement (LEE) core region and has a total length of 111 kb. About 43 kb upstream of the LEE core a gene cassette consisting of efa1/lifA gene and flanking IS elements suggests another putative transposon within the PAI(IRW1374). In addition, the ent gene, encoding a Shigella ShET-2 enterotoxin homologue, is present about 57 kb upstream of the LEE core. This PAI is therefore a complex assembly of various virulence determinants including the efa1/lifA and the ent gene resembling O157:H7 PAI OI-122/SpLE3 as well as the LEE core region. An integrase gene on the very left end of PAI I(Rw1374) is disrupted by an IS629 homologue. In an attempt to mobilize the LEE core we performed conjugation, transformation and transduction experiments. We were, however, unable to mobilize the whole or even single regions of PAI I(RW1374). Comparative studies with other strains of serotype O103:H2 isolated from humans, bovines and food showed that they all harbored a similar phe V-inserted PAI including the virulence genes ent and lifA/efa1 as well as the large virulence-associated plasmid encoding the EHEC hemolysin. This combination of several virulence factors confirms the complex virulence of O103:H2 EHEC and may at least partly explain the high virulence of this EHEC serotype in humans.

This review summarizes our current knowledge and models of appearance and dissemination of the locus of enterocyte effacement (LEE) within Escherichia coli phylogenetic lineages. The LEE is a pathogenicity island (PAI) required for attaching and effacing (A/E) lesion formation induced on epithelial cells of humans and animals by enteropathogenic and numerous enterohemorrhagic E. coli strains as well as other related bacteria. The LEE encodes a type III secretion system, an adhesin (intimin) responsible for the intimate attachment of the bacteria to the cell and a number of secreted proteins involved in signal transduction events. It has been shown that the LEE varies in size from 36 to 111 kb, depending on what E. coli lineages carrying that PAI. Three tRNA genes are known as LEE integration sites selC, pheU and pheV, the latter two are identical in sequence. Beneath its functional role, intimin is considered a phylogenetic marker of the LEE. Currently, 14 different intimin types have been described, designated alpha through ksi. Beta intimin-carrying LEEs moved within certain E. coli lineages from the pheU tRNA gene into the pheV tRNA gene. Moreover, as a result of the typing of multiple LEE core regions, the appearance of two different LEE cores indicates an import of the LEE within E. coli at least two times.

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides, is an important livestock disease in Africa. The current control measures rely on a vaccine with limited efficacy and occasional severe side effects. Knowledge of the protective arms of immunity involved in this disease will be beneficial for the development of an improved vaccine. In previous studies on cattle infected with M. mycoides subsp. mycoides, a correlation was detected between the levels of mycoplasma-specific IFN-γ-secreting CD4+ T lymphocytes and reduced clinical signs. However, no cause and effect has been established, and the role of such cells and of protective responses acquired during a primary infection is not known. We investigated the role of CD4+ T lymphocytes in CBPP by comparing disease patterns and post mortem findings between CD4+ T cell depleted and non-depleted cattle. The depletion was carried out using several injections of BoCD4 specific murine monoclonal antibody on day 6 after experimental endotracheal infection with the strain Afadé. All cattle were monitored clinically daily and sacrificed 28-30 days post-infection. Statistically significant but small differences were observed in the mortality rate between the depleted and non-depleted animals. However, no differences in clinical parameters (fever, signs of respiratory distress) and pathological lesions were observed, despite elimination of CD4+ T cells for more than a week. The slightly higher mortality in the depleted group suggests a minor role of CD4+ T cells in control of CBPP.