Identification, Characterization and Purification of MSC_0265, a Potential Immunogenic Antigen Homologue of Mycoplasma mycoides subsp. mycoides in Mycoplasma capricolum subsp. capripneumoniae

In silico identification and characterization of vaccine antigens has opened up new frontiers in the field of reverse vaccinology to mitigate the effects of livestock diseases by development of new subunit vaccines. This study aims to characterize, express and purify MSC_0265 for eventual use in immunoassays and inoculation in goats. Mycoplasma mycoides subs. mycoides (Mmm) and Mycoplasma capricolum capripneumoniae (Mccp) are similar pathogens on the genomic level and are the causative agents of Contagious Bovine Pleuropneumonia (CBPP) in cattle and Contagious Caprine Pleuropneumonia (CCPP) in goats respectively. In this study, BLAST was used to identify the homology of MSC_0265 in Mycoplasma capricolum capripneumoniae genome and the protein it is similar to. Characterization of MSC_0265 was also done using I-TASSER to predict secondary structure, solvent accessibility, normalised B-factor, 3D models and function. With cut off points of 0.0 for E-value, 100% for Query coverage and 90% for Identity, MSC_0265 a pyruvate dehydrogenase enzyme gave a high homology score on tBLASTn and BLASTp. It had earlier been cloned in pGS21a vector before proceeding with expression and purification of the His-tagged protein by Ni-NTA affinity chromatography. This study identified the homologue of MSC_0265 as protein WP_029333261.1 in the Mycoplasma capricolum capripneumoniae genome (Accession NZ_LN515398.1) using tBLASTn and BLASTp. Additionally, MSC_0265 was characterized and its optimal expression profile and estimated molecular weight verified

Multiple evolutionary origins of Trypanosoma evansi in Kenya

Trypanosoma evansi is the parasite causing surra, a form of trypanosomiasis in camels and other livestock, and a serious economic burden in Kenya and many other parts of the world. Trypanosoma evansi transmission can be sustained mechanically by tabanid and Stomoxys biting flies, whereas the closely related African trypanosomes T. brucei brucei and T. b. rhodesiense require cyclical development in tsetse flies (genus Glossina) for transmission. In this study, we investigated the evolutionary origins of T. evansi. We used 15 polymorphic microsatellites to quantify levels and patterns of genetic diversity among 41 T. evansi isolates and 66 isolates of T. b. brucei (n = 51) and T. b. rhodesiense (n = 15), including many from Kenya, a region where T. evansi may have evolved from T. brucei. We found that T. evansi strains belong to at least two distinct T. brucei genetic units and contain genetic diversity that is similar to that in T. brucei strains. Results indicated that the 41 T. evansi isolates originated from multiple T. brucei strains from different genetic backgrounds, implying independent origins of T. evansi from T. brucei strains. This surprising finding further suggested that the acquisition of the ability of T. evansi to be transmitted mechanically, and thus the ability to escape the obligate link with the African tsetse fly vector, has occurred repeatedly. These findings, if confirmed, have epidemiological implications, as T. brucei strains from different genetic backgrounds can become either causative agents of a dangerous, cosmopolitan livestock disease or of a lethal human disease, like for T. b. rhodesiense

Phase II Evaluation of Sensitivity and Specificity of PCR and NASBA Followed by Oligochromatography for Diagnosis of Human African Trypanosomiasis in Clinical Samples from D.R. Congo and Uganda

Diagnosis plays a central role in the control of human African trypanosomiasis (HAT) whose mainstay in disease control is chemotherapy. However, accurate diagnosis is hampered by the absence of sensitive techniques for parasite detection. Without concentrating the blood, detection thresholds can be as high as 10,000 trypanosomes per milliliter of blood. The polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA) are promising molecular diagnostics that generally yield high sensitivity and could improve case detection. Recently, these two tests were coupled to oligochromatography (OC) for simplified and standardized detection of amplified products, eliminating the need for electrophoresis. In this study, we evaluated the diagnostic accuracy of these two novel tests on blood specimens from HAT patients and healthy endemic controls from D.R. Congo and Uganda. Both tests exhibited good sensitivity and specificity compared to the current diagnostic tests and may be valuable tools for sensitive and specific parasite detection in clinical specimens. These standardized molecular test formats open avenues for improved case detection, particularly in epidemiological studies and in disease diagnosis at reference centres

Murine Models for Trypanosoma brucei gambiense Disease Progression—From Silent to Chronic Infections and Early Brain Tropism

Trypanosoma brucei gambiense is responsible for more than 90% of reported cases of human African trypanosomosis (HAT). Infection can last for months or even years without major signs or symptoms of infection, but if left untreated, sleeping sickness is always fatal. In the present study, different T. b. gambiense field isolates from the cerebrospinal fluid of patients with HAT were adapted to growth in vitro. These isolates belong to the homogeneous Group 1 of T. b. gambiense, which is known to induce a chronic infection in humans. In spite of this, these isolates induced infections ranging from chronic to silent in mice, with variations in parasitaemia, mouse lifespan, their ability to invade the CNS and to elicit specific immune responses. In addition, during infection, an unexpected early tropism for the brain as well as the spleen and lungs was observed using bioluminescence analysis. The murine models presented in this work provide new insights into our understanding of HAT and allow further studies of parasite tropism during infection, which will be very useful for the treatment and the diagnosis of the disease

Performance of conventional PCR for detection of Mycobacterium tuberculosis in mouthwashes

BACKGROUNDLack of rapid, sensitive, and affordable diagnostics has greatly hampered tuberculosis control efforts in countries with high prevalence of human immunodeficiency virus (HIV) infection and anti-tuberculosis drug resistance. Although sputum smear microscopy remains the principal tool for diagnosing active Pulmonary tuberculosis, its sensitivity is quite low. The impact of sputum culture and drug susceptibility testing is limited by the long duration and complexity of the laboratory processes. Additional diagnostic challenges posed by extra-pulmonary tuberculosis, pediatric tuberculosis, and latent tuberculosis infection. M. tuberculosis PCR amplification in mouthwashes was compared with existing methods for diagnosis of tuberculosis.MATERIALS AND METHODSThis study was carried out at Mbagathi Hospital, Nairobi, between January 2016 and December 2018. During the study period, all adult patients of either sex referred to the Mbagathi Hospital TB laboratory with clinical features suggestive of tuberculosis were recruited into the study. Mouthwashes were collected through rinsing with normal saline. Mouthwash results were compared with that of reference standard culture, Ziehl–Neelsen (ZN) smear microscopy the GeneXpert.RESULTSOf the 300 patients that fitted the study inclusion criteria, acceptable specimen samples were obtained from 210 patients whereby 165 patients whose cultures were read as either positive or negative had their results analyzed.70 (42.4%) patients were both culture and ZN smear-positive whereas 87(52.7%) were both culture and ZN smear negative.7(4.2%) patients were culture negative but ZN positive whereas 1(0.6%) was culture-positive but ZN smear negative.69(41.8%) patients were positive for both culture and PCR whereas 80(48.4%) were negative for both cultures and PCR.14 (8.4%) patients were, however, negative for culture but PCR positive.2(2.4%) of the patients were culture-positive but PCR negative.66 (40.7%) of the patients tested positive for both culture and GeneXpert whereas 87(53.7%) were both culture and GeneXpert negative. 2(1.2%) of the patients were culture negative but positive for GeneXpert and lastly,7 (4.3%) of the patients were culture-positive but GeneXpert negative. 45(27%) of the patients had their cultures contaminated. The test performances were as follows:100%,94%,92% and 94% for culture,90.1%,99%,90% and 91% for ZN smear, 83%,97%,81% and 83% for PCR and 97.1%,92%,88% and 90.1% for the GeneXpert respectively.CONCLUSIONSPCR test accurately and rapidly detected M. tuberculosis - specific DNA sequences of small numbers of mycobacteria in mouthwashes and was easily manipulable. Further refinements of the test may improve the diagnosis of tuberculosis in resource-constrained countries

Genome and phylogenetic analyses of Trypanosoma evansi reveal extensive similarity to T. brucei and multiple independent origins for dyskinetoplasty.

Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA). In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS) having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs) were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F1FO-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei