Respiratory explants as a model to investigate early events of contagious bovine pleuropneumonia infection.

Contagious bovine pleuropneumonia (CBPP) is a severe disease caused by Mycoplasma mycoides subsp. mycoides (Mmm). Knowledge on CBPP pathogenesis is fragmented and hampered by the limited availability of laboratory animal and in vitro models of investigation. The purpose of the present study is to assess respiratory explants as useful tools to study the early stages of CBPP. Explants were obtained from trachea, bronchi and lungs of slaughtered cattle, tested negative for Mycoplasma spp. and for the major bacterial and viral respiratory pathogens. The interaction of Mmm with explant cells was studied by immunohistochemistry (IHC), double-labelling indirect immunofluorescence (DLIIF) and laser scanning confocal microscopy (LSCM). Mmm capability to survive and proliferate within the explants was evaluated by standard microbiological procedures. Finally, the putative cellular internalization of Mmm was further investigated by the gentamicin invasion assay. IHC and DLIIF indicated that Mmm can colonize explants, showing a marked tropism for lower airways. Specifically, Mmm was detected on/inside the bronchiolar and alveolar epithelial cells, the alveolar macrophages and the endothelial cells. The interaction between Mmm and explant cells was abolished by the pre-incubation of the pathogen with bovine anti-Mmm immune sera. Mmm was able to survive and proliferate in all tracheal, bronchial and lung explants, during the entire time course of the experiments. LSCM and gentamicin invasion assay both confirmed that Mmm can enter non-phagocytic host cells. Taken together, our data supports bovine respiratory explants as a promising tool to investigate CBPP, alternative to cattle experimental infection.

Minimal Cells-Real and Imagined.

A minimal cell is one whose genome only encodes the minimal set of genes necessary for the cell to survive. Scientific reductionism postulates the best way to learn the first principles of cellular biology would be to use a minimal cell in which the functions of all genes and components are understood. The genes in a minimal cell are, by definition, essential. In 2016, synthesis of a genome comprised of only the set of essential and quasi-essential genes encoded by the bacterium Mycoplasma mycoides created a near-minimal bacterial cell. This organism performs the cellular functions common to all organisms. It replicates DNA, transcribes RNA, translates proteins, undergoes cell division, and little else. In this review, we examine this organism and contrast it with other bacteria that have been used as surrogates for a minimal cell.

Qualitative and quantitative impacts assessment of contagious bovine pleuropneumonia in Fulani pastoral herds of North-central Nigeria: The associated socio-cultural factors.

Contagious bovine pleuropneumonia is one of the most important trans-boundary disease affecting Fulani cattle herds of Nigeria and whose control is urgently needed. A Participatory Epidemiology approach and cross-sectional study were concurrently conducted to investigate qualitative and quantitative impacts of CBPP, respectively and associated socio-cultural factors that influenced exposure of Fulani nomadic pastoral communities to its risk in Niger State, North-central Nigeria between January and December 2013. A total of nine pastoral communities were purposively selected for qualitative impact assessment using Participatory Rural Appraisal tools, while 765 cattle randomly sampled from 125 purposively selected nomadic herds were analyzed using c-ELISA. Data on socio-cultural characteristics were collected using structured questionnaires administered on nomadic herd owners of the 125 selected herds. Kendall's Coefficient of Concordance W statistics and OpenEpi 2.3 were used for statistical analyses. Pastoralists' dependent factors associated with their socio-cultural activities were tested using Chisquare tests and likelihood backward logistic regressions. The mean proportional piles (relative qualitative impact) of CBPP was 12.6%, and nomads agreement on this impact was strong (W=0.6855) and statistically significant (P<0.001). This was validated by 16.2% (95% CI: 13.7, 19.0) sero-positive (quantitative impact). Highest sero-prevalence of 25.3% was observed in Northern agro-ecological zone, while lowest of 6.2% was in Eastern zone. Pastoralists in the age groups 51-60 and 61-70 years were more likely (OR 13.07; 95% CI: 3.21, 53.12 and OR 7.10; 95% CI: 1.77, 28.33, respectively) to have satisfactory information/awareness on CBPP and lowland transhumance pastoralists were more likely (OR 5.21; 95% CI: 2.01, 13.54) to have satisfactory information. Socio-cultural activities of extensive husbandry system was six times more likely (OR 5.79; 95% CI: 2.55, 13.13) to be satisfactory practice that influenced CBPP occurrence in herds, while culture of borrowing and loaning of cattle was twenty times more likely (OR 19.94; 95% CI: 6.36, 62.48) to be satisfactory practice that influenced CBPP occurrence in herds. Also, sharing a water source that caused concentration of stocks in one point was fifty three times more likely (OR 53.08; 95% CI: 14.91, 189.00) to be satisfactory practice that influenced occurrence of the disease in herds. This study highlighted the critical gap that exists in terms of significant influence of socio-cultural factors on CBPP occurrence in pastoral herds in Nigeria. Thus, CBPP surveillance, control and prevention programs that take these factors into consideration will be beneficial to the livestock industry in Nigeria, and indeed Africa.

P19 contributes to Mycoplasma mycoides subsp. mycoides adhesion to EBL cells.

Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP). The virulent Mmm Ben-1 strain was isolated from the lung of a CBPP-infected cow in China in the 1950s. To attenuate the virulence of the Ben-1 strain and preserve its protective ability, the isolate was re-isolated after inoculation into the testicles of rabbits and into the rabbit thorax. As a result, after the subsequent isolates were continuously passaged 468 times in rabbits, its pathogenicity to cattle decreased. However, the molecular mechanisms leading to attenuation of the Mmm Ben-1 remain unknown. We compared the entire genomes of the Ben-1 strain and the 468 th generation strain passaged in rabbits (Ben-468) and discovered that a putative protein gene named p19 was absent from the Ben-468 strain. The p19 gene was cloned and expressed in Escherichia coli to obtain recombinant P19 (rP19). Western blot analysis demonstrated that the P19 protein is detected in the cell-membrane fraction, the cell-soluble cytosolic fraction and whole-cell lysate of the Mmm Ben-1 strain. The rP19 can interact with international standard serum against CBPP. Immunostaining visualised via confocal laser scanning microscopy indicated that P19 is able to adhere to embryonic bovine lung (EBL) cells, and this finding was also confirmed by a sandwich ELISA. We also found that anti-rP19 serum could inhibit the adhesion of the Mmm Ben-1 total proteins to EBL cells.

Galactofuranose in Mycoplasma mycoides is important for membrane integrity and conceals adhesins but does not contribute to serum resistance.

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 catalysing 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 ß-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.

Quantitative risk assessment of entry of contagious bovine pleuropneumonia through live cattle imported from northwestern Ethiopia.

Contagious bovine pleuropneumonia (CBPP) is a highly contagious bacterial disease of cattle caused by Mycoplasma mycoides subspecies mycoides small colony (SC) bovine biotype (MmmSC). It has been eradicated from many countries; however, the disease persists in many parts of Africa and Asia. CBPP is one of the major trade-restricting diseases of cattle in Ethiopia. In this quantitative risk assessment the OIE concept of zoning was adopted to assess the entry of CBPP into an importing country when up to 280,000 live cattle are exported every year from the northwestern proposed disease free zone (DFZ) of Ethiopia. To estimate the level of risk, a six-tiered risk pathway (scenario tree) was developed, evidences collected and equations generated. The probability of occurrence of the hazard at each node was modelled as a probability distribution using Monte Carlo simulation (@RISK software) at 10,000 iterations to account for uncertainty and variability. The uncertainty and variability of data points surrounding the risk estimate were further quantified by sensitivity analysis. In this study a single animal destined for export from the northwestern DFZ of Ethiopia has a CBPP infection probability of 4.76×10(-6) (95% CI=7.25×10(-8) 1.92×10(-5)). The probability that at least one infected animal enters an importing country in one year is 0.53 (90% CI=0.042-0.97). The expected number of CBPP infected animals exported any given year is 1.28 (95% CI=0.021-5.42). According to the risk estimate, an average of 2.73×10(6) animals (90% CI=10,674-5.9×10(6)) must be exported to get the first infected case. By this account it would, on average, take 10.15 years (90% CI=0.24-23.18) for the first infected animal to be included in the consignment. Sensitivity analysis revealed that prevalence and vaccination had the highest impact on the uncertainty and variability of the overall risk.

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.

Cyto-adherence of Mycoplasma mycoides subsp. mycoides to bovine lung epithelial cells.

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.

Survival capacity of Mycoplasma agalactiae and Mycoplasma mycoides subsp capri in the diluted semen of goat bucks and their effects on sperm quality.

This study examines the viability of Mycoplasma agalactiae (Ma) and Mycoplasma mycoides subsp capri (Mmc) during 150 minutes of incubation at 37 °C in contaminated diluted semen (DS) doses. The effects of the presence of both microorganisms on sperm viability, motility, and morphology were also examined. In a second experiment, the viability of Ma and its effects on sperm viability were determined in ejaculate samples and skimmed milk semen extender samples. Ma and Mmc were able to survive in DS at concentrations considered infectious, and no significant differences in mean concentrations were detected (7.1 log colony-forming units [CFU]/mL). However, initial concentration of Ma declined (P < 0.05) from 7.5 to 6.9 log CFU/mL and Mmc declined (P < 0.05) from 7.7 to 7.1 log CFU/mL after incubation. Conversely, ejaculate concentrations of Ma increased significantly (from 7.1 to 7.4 log CFU/mL, P < 0.05). These observations suggest that the natural breeding medium is more suitable for Ma than the medium used for artificial insemination (AI). The presence of Mmc slightly reduced sperm viability in the DS (from 21.7% to 16.6%, P < 0.05). The absence of major effects on sperm quality could lead to the unnoticed use of semen contaminated with Ma and Mmc for AI. As both bacteria were able to survive the conditions of ejaculates and semen doses, these findings suggest a risk of venereal transmission of contagious agalactia and support the use of mycoplasma-free semen samples for (AI).

Comparison of in vivo and in vitro properties of capsulated and noncapsulated variants of Mycoplasma mycoides subsp. mycoides strain Afadé: a potential new insight into the biology of contagious bovine pleuropneumonia.

Mycoplasma mycoides subsp. mycoides (Mmm) strain Afadé had previously been shown to undergo spontaneous phase variations between an opaque capsulated variant and a translucent (TR) variant devoid of a capsule but able to secrete cell-free exopolysaccharides. This phase variation is associated with an ON/OFF genetic switch in a glucose permease gene. In this study, in vivo and in vitro assays were conducted to compare the virulence of the two variants and their abilities to resist host defence. Capsulated variants were shown, in a mouse model, to induce longer bacteraemia that was correlated with better serum resistance in vitro. In contrast, TR variants displayed better ability to adhere to an inert support, linked to the absence of a capsule, changes in cell surface hydrophobicity and increased resistance to antimicrobial peptide and hydrogen peroxide. The switch from one variant population to another, which was observed both in vivo and in vitro under stress conditions, is further discussed as a means for Mmm to modulate its interactions with animal hosts during different stages of the disease.

Estimation of impact of contagious bovine pleuropneumonia on pastoralists in Kenya.

Contagious bovine pleuropneumonia (CBPP) is an infectious disease which impacts cattle production in sub-Saharan Africa. To adequately allocate resources for its control, there is a need to assess its impact on cattle producers. The present study estimated the impact of CBPP on pastoralists through analysis of various strategies employed for its control in cattle herds including: preventive vaccination, antimicrobial treatment, slaughter of clinical cases and other combinations of these control strategies. The assessment was based on a loss-expenditure frontier framework to identify a control strategy with minimum cost from both expenditures on control strategies and output losses due to mortalities, reduced milk yield, reduced weight gain and reduced fertility rate. The analysis was undertaken in a stochastic spreadsheet model. The control strategy with minimum cost per herd was preventive vaccination with an estimated cost of US$ 193 (90% CI; 170-215) per 100 cows per year, while slaughter of clinical cases had an estimated cost of US$ 912 (90% CI; 775-1055) per 100 cows per year. The impact of CBPP to the nation was estimated at US$ 7.6 (90% CI; 6.5-8.7) million per year. Yet, if all pastoralists whose cattle are at high risk of infection adopted preventive vaccination, the aggregate national impact would be US$ 3.3 (90% CI; 2.9-3.7) million per year, with savings amounting to US$ 4.3 million through reallocation of control expenditures. The analysis predicted that control of CBPP in Kenya is profitable through preventive vaccination. However, further research is recommended for the technical and financial feasibility of implementing a vaccine delivery system in pastoral areas where CBPP is endemic.

Private sector involvement in the control of contagious bovine pleuropneumonia (CBPP) in the Kazungula district of Zambia benefitted the community and the control strategy.

Contagious bovine pleuropneumonia (CBPP) is a disease of economic importance that is widely distributed in sub-Saharan African and contributes significantly to cattle morbidity and mortality. Lack of resources to implement eradication measures has led to the disease becoming endemic in most areas in sub-Saharan Africa where governments have little resources and the majority of the people are poor. Usually, control and eradication of such diseases as CBPP is treated as a public good by governments and to achieve this, governments are usually assisted by nongovernment organisations, bilateral government programmes and international donors. The private sector, which usually is companies that run businesses to make profit, although not very well established in sub-Saharan Africa could play a big role in the eradication of CBPP in the region. This could play a dual role of promoting investment and also eradicate livestock diseases which have proved a menace in the livestock sector. This paper highlights the role played by the private sector in the control of CBPP in Zambia.

Survival of Mycoplasma agalactiae and Mycoplasma mycoides subspecies capri in heat treated goat colostrum.

The viability of Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri (Mmc) was assessed in goat colostrum treated at different temperatures. Samples of colostrum were inoculated with reference strains of M. agalactiae (PG2) and Mmc (PG3) and heated at 56°C or 60°C for 0, 30, 60, 90 or 120 min. Viable colonies of M. agalactiae were recovered after all treatments and there was a significant reduction in the concentration of viable M. agalactiae after 30 min at 56°C and 60°C. No viable colonies of Mmc were observed after 60 min at 60°C.

Anatomic location of Mycoplasma mycoides subsp. capri and Mycoplasma agalactiae in naturally infected goat male auricular carriers.

This study sought to determine whether male goat auricular carriers of mycoplasmas known to cause contagious agalactia could harbour these microorganisms at anatomical sites other than the ears. A microbiological study was conducted in 6 naturally infected bucks that had been diagnosed as chronic auricular asymptomatic carriers of Mycoplasma (M.) mycoides subsp. capri (Mmc) more than one year previously. To detect mycoplasmas, cultures and PCR were performed on 46 samples taken from each goat from the cardio-respiratory, digestive, nervous, lymph and genitourinary systems and several joints. Of a total of 274 samples analyzed, 28 were positive for mycoplasmas (10.1%): Mmc was detected in 17 (6.1%), Mycoplasma (M.) agalactiae in 12 (4.3%) and both microorganisms were identified in one of the samples. In all 6 goats, mixed infection was observed despite none being auricular carriers of M. agalactiae. Mycoplasma spp. were identified at 15 different sites; the most frequent sites being the joints (31.2%, 5 positive samples), lymph nodes (25%, 4 positive samples) and respiratory tract (25%, 4 positive samples). Positive results were also obtained in three brain tissue (18.7%), two cardiac tissue (12.5%) and one ileum, urethra, testicle and bulbourethral gland (6.25%) samples. The histopathological findings may suggest the presence of mild chronic conditions in some of the organs where the bacteria were found. Our findings reveal for the first time the capacity of Mmc and M. agalactiae to colonize several other organ systems in chronically naturally infected auricular carriers, possibly representing an added risk factor for the spread of these microorganisms. In the case of M. agalactiae, colonization seemed to be independent of the animal's auricular carrier state.

Synthetic biology: now that we're creators, what should we create?

A 'synthetic' microbe has been created by introducing the artificially produced genome of one species into the cytoplasm of another. The technology allows the introduction of easily transferable adaptive units, as well as sets of genes that have likely never been transferred successfully.

Creation of a bacterial cell controlled by a chemically synthesized genome.

We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication.

Comparison of culture and PCR to detect Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri in ear swabs taken from goats.

This study was designed to evaluate the validity of PCR for the direct detection of Mycoplasma (M.) agalactiae and Mycoplasma mycoides subsp. capri (Mmc), as the two species most frequently causing contagious agalactia (CA) in goats. The PCR method was compared with the traditional culture technique to determine which method was most efficient at identifying all auricular carriers present in herds. The samples analyzed were 307 ear swabs taken from goats reared in a CA endemic area. We assessed the validity of each technique to detect each species and agreement between both methods. For each species, the result was taken as true-positive when at least one of the two tests was positive. Of the swabs tested, 246 were scored positive by PCR (235 and 11 for Mmc and M. agalactiae, respectively) and 117 showed a positive culture result (113 for Mmc and 4 for M. agalactiae). 133 of the PCR-positive samples (124 and 9 for Mmc and M. agalactiae, respectively) yielded negative culture results and 4 culture-positive samples tested negative using PCR (2 for each species). Sensitivity and negative predictive values for PCR were 84.62 and 99.32 (for M. agalactiae) and 99.16 and 97.22% (for Mmc) respectively, and for culture were 30.77 and 97.03 (for M. agalactiae) and 47.08 and 36.08% (for Mmc), respectively. PCR proved to be a rapid and sensitive method for the detection of mycoplasmas in the external ear of asymptomatic carriers. Tools such as this are needed to adopt efficient control measures against CA.

Phage display-based identification and potential diagnostic application of novel antigens from Mycoplasma mycoides subsp. mycoides small colony type.

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.

Experimental infection of goats with an unusual strain of Mycoplasma mycoides subsp. capri isolated in Jordan.

Goats were infected experimentally with a mycoplasma (the "Irbid" strain) isolated previously from a goat with contagious agalactia in northern Jordan. The strain was unusual in that, although it had been identified by molecular methods as Mycoplasma mycoides subsp. mycoides LC/Mycoplasma mycoides subsp. capri, it showed no inhibition of growth by any of the hyperimmune rabbit antisera conventionally used to speciate members of the Mycoplasma mycoides cluster. Animals were infected either intratracheally or by aerosol and placed "in-contact" with other goats. After 2 weeks, those infected intratracheally became febrile, showing a nasal discharge and slight conjunctivitis, followed a week later by respiratory distress and polyarthritis; lesions seen at necropsy included coagulative necrotic pneumonia, fibrinous pleurisy with pleural exudate, and inflammatory exudates, necrosis and fibrosis in the joints. Animals infected by aerosol showed much milder clinical signs, including nasal discharge and occasional swollen joints. In the "in-contact" goats, seroconversion was first seen after 7 weeks, accompanied by coughing and laboured respiration; lesions in this group consisted of fibrinous pneumonia with focal areas of necrosis and abundant pleural exudate.