A TaqMan-based RT-qPCR assay for serotyping of Southern African territories (SAT) 2 strains of Foot-and-Mouth disease virus (FMDV) in Southern Africa.

OBJECTIVE: Determining the serotype of circulating virus strains is important in implementing effective vaccination. In this study, Foot-and-Mouth Disease (FMD) Southern African territory 2 (SAT2) specific primers and TaqMan probe were designed towards rapid SAT2 detection and serotyping. The primers were tested by endpoint reverse transcription (RT) polymerase chain reaction (PCR) and quantitative PCR (RT-qPCR) using the vaccine strain SAT2035. The SAT2 serotype-specific RT-qPCR assay was compared with currently used ELISA and VP1 sequencing using Cohen's kappa statistics. RESULTS: The primers yielded amplicons of band size 190 bp during endpoint RT-PCR. When coupled with the probe, the primers reaction efficiency was determined to be 99% with an r2 value of 0.994. The results show that the SAT2 assay has comparable performance to VP1 sequencing (k = 1) and a moderate degree of agreement with ELISA (k = 0.571). The data shows that the newly designed assay could be considered for serotyping of SAT2 strains. However, for this assay to be complete there is a need to design effective SAT1 and SAT3 primers and probes that can be multiplexed to target other serotypes that co-circulate within relevant FMD endemic pools. For future implementation of the assay there is also a need to increase the number of field samples towards validation of the assay.

Do microbes evade domestication? - Evaluating potential ferality among diastatic Saccharomyces cerevisiae.

Certain lineages of the wine, beer and bread yeast Saccharomyces cerevisiae have diastatic activity. They contain the chimeric gene STA1 that codes for an extracellular glucoamylase which enables the strains to degrade starch and dextrins. Beer contaminations by diastatic yeasts can be dangerous because they can cause super-attenuation due to the consumption of otherwise non-fermentable oligosaccharides, gushing and off-flavours. Given that diastatic yeasts can be used for beer fermentation it is important to understand the relationship between production and contaminant strains, their natural reservoirs and entry routes into the brewery. Here, we analyze real cases of contamination in a Portuguese craft brewery over a period of 18 months. By analyzing with whole genome sequencing several contaminants, we show that recurrent contaminations by diastatic yeasts are caused by environmental strains. Moreover, some beer contaminants were closely related to diastatic environmental strains isolated in Botswana. We observed the widespread presence of domestication signatures in diastatic strains. Moreover, the combined phylogeny of STA1 and its ancestor, SGA1, suggested a single STA1 origin, as ancient as the entire lineage of diastatic yeasts. Together, our results suggest that diastatic yeasts isolated in natural settings could be escaping from domestication settings and becoming feral.

Heartwater: A situation report of the Southern district of Botswana from 2017 to 2019.

Heartwater is a tick-borne haemoparasitic disease that can limit agro-business expansion in Botswana. It poses a threat to national food security due to low animal production as well as livestock morbidity and mortality. This report gives a snapshot view of heartwater in the Southern district of Botswana. Ixodid ticks parasitizing livestock in four Southern sub-districts of Botswana were collected and identified using morphological and molecular methods. A wide distribution of Amblyomma hebraeum in all four Southern sub-districts was revealed. The annual number of heartwater cases across the Southern district of Botswana was determined from veterinary clinical case reports and confirmed through Giemsa-stained brain smears. A concerning gradual annual increase in heartwater cases was shown in the Moshupa sub-district - a hardveld terrain with rock outcrops where the vector thrives. Goats were affected most (55%) by heartwater followed by sheep (37%) and then cattle (8%). Farmers were interviewed on the management of the heartwater burden within their respective sub-districts and they reported that their animals were affected by heartwater despite 17 out of the 27 farmers interviewed attempting to control vectors through acaricide use. The presented heartwater situation warrants further investigation of the prevalence of heartwater and the effectiveness of existing disease control interventions in the disease-endemic Southern district of Botswana.

Mycotoxins in khadi, A Traditional Non-Cereal Based Alcoholic Beverage of Botswana.

Mycotoxin contamination is a major food safety drawback towards the commercialization of food products. The commercialization of khadi, a popular fermented alcoholic beverage of Botswana necessitates the investigation of the presence of mycotoxins. Khadi brewing involves the uncontrolled and unstandardized spontaneous fermentation of sun-dried Grewia flava fruits, which could be a source of mycotoxin-producing filamentous fungi (molds). This study sought to investigate the presence of mycotoxins producing fungi and mycotoxins in 18 samples of khadi collected in Central and Northern Botswana. Periconia thailandica, Cladosporium cladosporioides, Aspergillus ochraceus, Phoma eupyrena, Setosphaeria turcica, Cladosporium sphaerospermum, Chaetomium longiciliata, and Flavodon ambrosius were identified in 10 out of 18 khadi samples. Mycotoxins were detected using the Myco-10 Randox Evidence Investigator biochip kit and confirmed using a UPLC-ESI-MS/MS. Mycotoxins such as paxilline, ochratoxin A, ergot alkaloids, aflatoxin G1/G2, and zearalenone were detected using the Myco-10 Randox Evidence Investigator biochip kit. The Myco-10 results revealed that the mycotoxins in the khadi samples were lower than the regulatory limits set by FDA or European Commission. Confirmation of results using an UPLC-ESI-MS/MS system involved confirming selected mycotoxins (AFB1, DON. ZEA, FB1, FB2, FB3, NIV, and OTA) from selected khadi samples (Palapye 1, Palapye 2, Letlhakane 2, Maun 3, Mmashoro 3, and Tonota 3). The UPLC results demonstrated that the aforementioned mycotoxins in the selected khadi samples were below the detection thresholds. The study shows that while fungal isolates were present, there is no to minimal danger/risk of exposure to toxic mycotoxins after consumption of khadi. Towards commercialization endeavors, the production process would necessitate minimal mycotoxin monitoring and product preservation but no detoxifying steps.

Functional Characterization of khadi Yeasts Isolates for Selection of Starter Cultures.

Yeasts play an important role in spontaneous fermentation of traditional alcoholic beverages. Our previous study revealed that a mixed-consortia of both Saccharomyces and non-Saccharomyces yeasts were responsible for fermentation of khadi, a popular, non-standardized traditional beverage with an immense potential for commercialization in Botswana. Functional characterization of isolated fermenting yeasts from mixed consortia is an indispensable step towards the selection of potential starter cultures for commercialization of khadi. In this study, we report the characterization of 13 khadi isolates for the presence of brewing-relevant phenotypes such as their fermentative capacity, ability to utilize a range of carbon sources and their ability to withstand brewing-associated stresses, as a principal step towards selection of starter cultures. Khadi isolates such as Saccharomyces cerevisiae, Saccharomycodes ludwigii and Candida ethanolica showed good brewing credentials but Lachancea fermentati emerged as the isolate with the best brewing attributes with a potential as a starter culture. However, we were then prompted to investigate the potential of L. fermentati to influence the fruity aromatic flavor, characteristic of khadi. The aroma components of 18 khadi samples were extracted using headspace solid phase micro-extraction (HSSPME) and identified using a GC-MS. We detected esters as the majority of volatile compounds in khadi, typical of the aromatic signature of both khadi and L. fermentati associated fermentations. This work shows that L. fermentati has potential for commercial production of khadi.

Acaricide resistance in Rhipicephalus appendiculatus ticks collected from different farming systems in Zimbabwe.

The larval packet test (LPT) was used to investigate resistance in Rhipicephalus appendiculatus ticks to the amidine (amitraz) and organophosphate (chlorfenvinphos) chemical acaricides in different farming systems in Mashonaland West Province in Zimbabwe. The study results showed emerging resistance (ER) to amitraz in small-scale and commercial farming systems. The tick populations in communal farming systems were susceptible to both acaricides. A similar trend was observed for chlorfenvinphos, where ER was observed in the small-scale farming systems compared to communal and commercial farms. Furthermore, resistance ratios (RR) were higher for amitraz as compared to chlorfenvinphos. This study suggests that management practices, acaricide formulations, applications on cattle, intensity, and frequency of use could be pre-disposing factors for the emerging resistance towards amitraz observed in R. appendiculatus ticks found in small-scale and commercial farming systems. Amitraz is the most common and frequently used acaricides in all farming systems, and hence, resistance is developing much faster than organophosphates. There is a need to investigate further acaricide use and management practices in Zimbabwe's cattle farming systems to develop practical strategies for prevention and management of tick acaricide resistance.

Complete Genome Sequence of an Antimicrobial-Producing Bacillus velezensis Sam8H1 Isolate from the Makgadikgadi Saltpans of Botswana.

The global antimicrobial drug resistance crisis requires urgency in searching for more effective broad-spectrum antimicrobial drugs. Here, we present a complete circular genome sequence and a plasmid of an antimicrobial-producing isolate, Bacillus velezensis strain Sam8H1, from the Makgadikgadi saltpans in Botswana. Bioinformatic analyses revealed 12 putative secondary metabolite biosynthetic gene clusters important for genome-guided drug discovery studies.

Isolation of serotype-specific antibodies against dengue virus non-structural protein 1 using phage display and application in a multiplexed serotyping assay.

The multidimensional nature of dengue virus (DENV) infections, which can be caused by four distinct serotypes of the virus, complicates the sensitivity of assays designed for the diagnosis of infection. Different viral markers can be optimally detected at different stages of infection. Of particular clinical importance is the early identification of infection, which is pivotal for disease management and the development of blood screening assays. Non-structural protein 1 (NS1) is an early surrogate marker of infection and its detection in serum coincides with detectable viraemia. The aim of this work was to isolate and characterise serotype-specific monoclonal antibodies that bind to NS1 for each of the four DENV serotypes. This was achieved using phage display and a subtractive biopanning strategy to direct the antibody selection towards serotype-specific epitopes. This antibody isolation strategy has advantages over immunisation techniques where it is difficult to avoid antibody responses to cross-reactive, immunodominant epitopes. Serotype specificity to recombinant antigen for each of the antibodies was confirmed by Enzyme Linked Immunosorbent Assay (ELISA) and Surface Plasmon Resonance. Confirmation of binding to native DENV NS1 was achieved using ELISA and immunofluorescence assay on DENV infected Vero cells. No cross-reactivity with Zika or Kunjin viruses was observed. A previously isolated pan-reactive antibody that binds to an immunodominant epitope was able to pair with each of the serotype-specific antibodies in a sandwich ELISA, indicating that the serotype specific antibodies bind to epitopes which are all spatially distinct from the immunodominant epitope. These antibodies were suitable for use in a multiplexed assay for simultaneous detection and serotyping of DENV NS1 in human serum. This work demonstrates that phage display coupled with novel biopanning strategies is a valuable in vitro methodology for isolation of binders that can discern amongst antigens with high homology for diagnostic applicability.

Computational Identification of Antibody Epitopes on the Dengue Virus NS1 Protein.

We have previously described a method to predict antigenic epitopes on proteins recognized by specific antibodies. Here we have applied this method to identify epitopes on the NS1 proteins of the four Dengue virus serotypes (DENV1-4) that are bound by a small panel of monoclonal antibodies 1H7.4, 1G5.3 and Gus2. Several epitope regions were predicted for these antibodies and these were found to reflect the experimentally observed reactivities. The known binding epitopes on DENV2 for the antibodies 1H7.4 and 1G5.3 were identified, revealing the reasons for the serotype specificity of 1H7.4 and 1G5.3, and the non-selectivity of Gus2. As DENV NS1 is critical for virus replication and a key vaccine candidate, epitope prediction will be valuable in designing appropriate vaccine control strategies. The ability to predict potential epitopes by computational methods significantly reduces the amount of experimental work required to screen peptide libraries for epitope mapping.