Polymorphism analysis of the apxIA gene of Actinobacillus pleuropneumoniae serovar 5 isolated in swine herds from Brazil.

The bacterium Actinobacillus pleuropneumoniae is the etiological agent of Contagious Porcine Pleuropneumonia, a disease responsible for economic losses in the swine industry worldwide. A. pleuropneumoniae is capable of producing proteinaceous exotoxins responsible for inducing hemorrhagic lesions, one of which is ApxI. Few studies have conducted an in-depth evaluation of polymorphisms of the nucleotides that make up the ApxI toxin gene. Here we analyze the polymorphisms of the apxIA gene region of A. pleuropneumoniae serovar 5 isolated from swine in different regions in Brazil and report the results of molecular sequencing and phylogenetic analysis. Analysis of the apxIA gene in 60 isolates revealed the presence of genetic diversity and variability. The polymorphisms in the nucleotide sequences determined the grouping of the Brazilian sequences and five more sequences from the GenBank database into 14 different haplotypes, which formed three main groups and revealed the presence of mutations in the nucleotide sequences. The estimation of selection pressures suggests the occurrence of genetic variations by positive selective pressure on A. pleuropneumoniae in large groups of animals in relatively small spaces. These conditions presumably favor the horizontal dissemination of apxIA gene mutations within bacterial populations with host reservoirs. As a result, the same serovar can demonstrate different antigenic capacities due to mutations in the apxIA gene. These alterations in sequences of the apxIA gene could occur in other areas of countries with intense swine production, which could lead to differences in the pathogenicity and immunogenicity of each serovar and have implications for the clinical status or diagnosis of A. pleuropneumoniae.

Mmm-derived lipid-associated membrane proteins activate IL-1ß production through the NF-κB pathway via TLR2, MyD88, and IRAK4.

Mycoplasma mycoides subsp.mycoides (Mmm) is a pathogen that causes pneumonia, otitis media, and arthritis in young calves. Its pathogenesis is attributed in part to excessive immune responses. Mmm-derived lipid-associated membrane proteins (LAMPs) are potent inducers of the host innate immune system; however, interactions between Mmm-derived LAMPs as pathogenic agents, toll-like receptors (TLRs), and the signaling pathways responsible for activating inflammation and nuclear factor (NF)-κB have not been fully elucidated. Here, we analyzed the expression kinetics of interleukin (IL)-1ß in Mmm-derived LAMP-stimulated embryonic bovine lung (EBL) cells and found that Mmm-derived LAMPs induced IL-1ß expression. Subcellular localization analysis revealed the nuclear translocation of the NF-κB p65 subunit after EBL cells were stimulated with Mmm-derived LAMPs. Furthermore, a specific inhibitor assay demonstrated that NF-κB is required for Mmm-derived LAMP-induced IL-1ß expression. Additionally, overexpression of TLR2, myeloid differentiation primary response gene 88 (MyD88), and IL-1 receptor-associated kinase 4 (IRAK4) increased IL-1ß expression during LAMP stimulation, and TLR2-neutralizing antibodies reduced IL-1ß expression in EBL cells during LAMP stimulation. Furthermore, LAMPs inhibited IL-1ß expression following transfection with dominant-negative MyD88 and IRAK4 variants. These results suggested that Mmm-derived LAMPs activate IL-1ß production through the NF-κB pathway via TLR2, MyD88, and IRAK4.

[Reemergence of contagious bovine pleuropneumonia in Senegal]. / Réémergence de la péripneumonie contagieuse bovine au Sénégal.

The authors have described an epizootic infection of contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides biotype Small Colony (MmmSC), that has affected Ndama bovine in Lounthy village, a locality based in Bala city in the Eastern part of Senegal, during the post-rainy season in November 2012. After the cessation of vaccination, a hotbed of suspicion of CBPP was identified on November 3rd 2012 in the village of Lounthy: out of the total of 98 cattle, 13 animals were sick and 5 of them died. These studies have been done according to clinical aspects, serological, bacteriological and molecular analysis of the samples. This reemergent disease will give new orientations for CBPP control in Senegal, where it was supposed the disease has been eradicated since 2005.

Disruption of the S41 peptidase gene in mycoplasma mycoides capri impacts proteome profile, H(2)O(2) production, and sensitivity to heat shock.

Members of the Mycoplasma mycoides cluster are among the most virulent of the mycoplasmas, causing worldwide economically significant diseases of cattle and goats. A distinguishing phenotype among the members of the cluster is the ability to degrade casein. The MMCAP2_0241 gene, an S41 peptidase, confers the proteolytic phenotype in Mycoplasma mycoides subsp. capri GM12. In order to determine the impact of disruption of the gene, we used differential proteome profiling to compare the M. mycoides subsp. capri wild type with a mutant lacking the proteolytic phenotype. Disruption of MMCAP2_0241 resulted in altered phenotypes reminiscent of M. mycoides subsp. mycoides SC and had significant impacts on the proteome profile of the microbe. The mutant exhibited increased production of hydrogen peroxide, decreased lactate dehydrogenase activity, and increased sensitivity to heat shock.

A lateral flow protein microarray for rapid determination of contagious bovine pleuropneumonia status in bovine serum.

Novel analytical methods for a next generation of diagnostic devices combine attributes from sensitive, accurate, fast, simple and multiplexed analysis methods. Here, we describe a possible contribution to these by the application of a lateral flow microarray where a panel of recombinant protein antigens was used to differentiate bovine serum samples in the context of the lung disease contagious bovine pleuropneumonia (CBPP). Lateral flow arrays were produced by attaching nitrocellulose onto microscopic slides and spotting of the recombinant proteins onto the membranes. The developed assay included evaluations of substrate matrix and detection reagents to allow for short assay times and convenient read-out options, and to yield a total assay time from sample application to data acquisition of less than ten minutes. It was found that healthy and disease-affected animals could be discriminated (AUC=97%), and we suggest that the use of an antigen panel in combination with the lateral flow device offers an emerging analytical tool towards a simplified but accurate on-site diagnosis.

First report on the molecular prevalence of Mycoplasma capricolum subspecies capripneumoniae (Mccp) in goats the cause of contagious caprine pleuropneumonia (CCPP) in Balochistan province of Pakistan.

Contagious caprine pleuropneumonia (CCPP) caused by Mycoplasma capricolum subspecies capripneumoniae (Mccp) is a disease of goats which causes high morbidity and mortality and is reported in many countries of the world. There are probably no reports on the molecular prevalence of Mccp, Mycoplasma capricolum subsp. capricolum (Mcc) and Mycoplasma putrefaciens (Mp) in Balochistan and any other part of Pakistan. Thirty goats (n = 30) with marked respiratory symptoms were selected and procured from forty goat flocks in Pishin district of Balochistan in 2008. The genomic deoxyribonucleic acid (DNA) from the lung samples (n = 30) of the slaughtered goats was purified and subjected to polymerase chain reaction (PCR) assays for the presence of Mycoplasma mycoides cluster members and Mp. The PCR-RFLP (restriction fragment length polymorphism) was also used to further confirm the Mccp. Of the thirty lung samples 17 (56.67%) were positive for the molecular prevalence of Mcc, Mccp and Mp. In total the molecular prevalence was observed as 17.65% for Mccp (n = 3), 70.59% for Mcc (n = 12) and 11.76% for Mp (n = 2). The RFLP profile has also validated the PCR results of Mccp by yielding two bands of 190 and 126 bp. The results of PCR-RFLP coupled with the presence of fibrinous pleuropneumonia and pleurisy during postmortem of goats (n = 3) strongly indicated the prevalence of CCPP in this part of world. Moreover the prevalence of Mcc and Mp is also alarming in the study area. We report for the very first time the molecular prevalence of Mcc, Mccp, and Mp in the lung tissues of goats in the Pishin district of Balochistan, Pakistan.

Specific real-time PCR assays for the detection and quantification of Mycoplasma mycoides subsp. mycoides SC and Mycoplasma capricolum subsp. capripneumoniae.

Contagious bovine pleuropneumonia and contagious caprine pleuropneumonia are two severe respiratory infections of ruminants due to infection by Mycoplasma mycoides subsp. mycoides SC (MmmSC) and Mycoplasma capricolum subsp. capripneumoniae (Mccp), respectively. They are included in the OIE list of notifiable diseases. Here we describe the development of rapid, sensitive, and specific real-time PCR assays for the detection and quantification of MmmSC and Mccp DNA. MmmSC PCR primers were designed after whole genome comparisons between the published sequence of MmmSC strain type PG1(T) and the sequence of an M. mycoides subsp. mycoides large colony strain. For Mccp, previously published conventional PCR primers were applied. SYBR green was used as a detection agent for both assays. The assays specifically detected the targeted species in both cultures and clinical samples, and no cross-amplifications were obtained from either heterologous mycoplasma strain cultures or European field samples. The sensitivity of these new assays was estimated at 3-80 colony forming units per reaction and 4-80fg of DNA, representing a 2-3log increase in sensitivity compared to established conventional PCR tests. These new real-time PCR assays will be invaluable for application in various fields such as direct detection in diagnostic laboratories.

Identification and characterization of IS1296 in Mycoplasma mycoides subsp. mycoides SC and presence in related mycoplasmas.

IS1296, a new insertion sequence belonging to the IS3 family of insertion elements has been identified in Mycoplasma mycoides subsp. mycoides (Mmm) biotype small colony (SC), the agent of contagious bovine pleuropneumonia (CBPP). IS1296 is 1485-bp long and has 30-bp inverted repeats. It contains two open reading frames, ORFA and ORFB, which show significant similarities to the ORFs which encode the transposase function of IS elements of the IS3 family, in particular IS150 of Escherichia coli. IS1296 is present in 19 copies in Mmm SC-type strain PG1 and in 18 copies in a recently isolated field strain L2. It seems to transpose at low frequency in Mmm SC. IS1296 is also present in 5 copies in Mmm biotype large colony (LC)-type strain Y-goat, and in two copies in Mycoplasma sp. 'bovine group 7' reference strain PG50. It is, however, not present in other species of the 'mycoides cluster' or other closely related Mycoplasma sp. of ruminants.