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Mycotoxin contamination is a major food safety drawback towards the commercialization of food products. The commercialization of

Yeasts play an important role in spontaneous fermentation of traditional alcoholic beverages. Our previous study revealed that a mixed-consortia of both

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.

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.

BACKGROUNDHeartwater is a tick-borne, haemoparasitic disease that has the potential to limit the growth of agro-businesses in Botswana. This threat to national food security is attributed to low production and mortality of livestock in subsistence farming of small stock than in commercial farms. This study gives a view of the epidemiological burden of heartwater in Botswana. METHODSFeeding ticks on livestock kept in four Southern sub-districts of Botswana were identified through morphological and molecular analysis. Farmers were interviewed on the management of the heartwater burden within their respective sub-districts. Veterinary clinical case reports were collected to assess the frequency of cases across the Southern District of Botswana. RESULTSOutcomes of the study revealed; a wide distribution of Amblyomma hebraeum in all four studied sub-districts, gradual annual increase in heartwater cases in Moshupa sub-district and indication of increased impact on indigenous caprine species despite the effort to control vectors by 63% of interviewed farmers. CONCLUSIONAmblyomma hebraeum, the principal vector of Ehrlichia ruminantium in Southern Africa is widely distributed in the Southern District of Botswana and thus poses a challenge to livestock productivity in this district and, perhaps, elsewhere in the country where the tick infests livestock. There is therefore a need to re-examine the existing tick control interventions with an intention to reduce the heartwater disease burden particularly in sheep and goats.

Khadi is a popular traditional alcoholic beverage in rural households in Botswana. The product is produced by fermentation of ripened sun-dried Grewia flava (Malvaceae) fruits supplemented with brown table sugar. Despite its popularity, its growing consumer acceptance, its potential nutritional value, and its contribution to the socio-economic lifestyle of Botswana, the production process remains non-standardized. Non-standardized production processes lead to discrepancies in product quality and safety as well as varying shelf life. Identification of unknown fermentative microorganisms of khadi is an important step towards standardization of its brewing process for entrance into commercial markets. The aim of this study was to isolate and identify bacteria and yeasts responsible for fermentation of khadi. Yeasts and bacteria harbored in 18 khadi samples from 18 brewers in central and northern Botswana were investigated using classic culture-dependent techniques and DNA sequencing methods. Additionally, we used the same techniques to investigate the presence of bacteria and yeasts on six batches of ripened-dried G. flava fruits used for production of the sampled brews. Our results revealed that Saccharomyces cerevisiae closely related to a commercial baker’s yeast strain sold locally was the most predominant yeast species in khadi suggesting a possible non-spontaneous brewing process. However, we also detected diverse non-Saccharomyces yeasts, which are not available commercially in retail shops in Botswana. This suggests that spontaneous fermentation is partially responsible for fermentation of khadi. This study, presenting the first microbiological characterization of a prominent traditional alcoholic beverage in Botswana, is vital for development of starter cultures for the production of a consistent product towards the commercialization of khadi.

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.

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.

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.

Dengue virus (DENV) infections are a significant public health burden in tropical and sub-tropical regions of the world. Infections are caused by four different but antigenically related viruses which result in four DENV serotypes. The multifaceted nature of DENV pathogenesis hinders the sensitivity of assays designed for the diagnosis of infection. Different markers can be optimally detected at different stages of infection. Of particular clinical importance is the identification of acute viremia during the febrile phase of infection which is pivotal for management of infection. Non-structural protein 1 (NS1) has been identified as a good early surrogate marker of infection with possible applications in epidemiological surveillance and the development of blood screening assays. This contribution is towards using serotype-specificity to achieve specific and more sensitive diagnostic detection of DENV NS1. The general aim of this work is to isolate immune-reagents that can be used to develop an assay with improved sensitivity of DENV NS1 detection in a diagnostic setting. In this work, we sought to isolate serotype-specific antibodies that discern discreet antigenic differences in NS1 from each DENV serotype. Additionally, we also sought to isolate a pairing antibody that recognises NS1 from all four DENV serotypes (pan-reactive) for tandem capture of the DENV NS1. To achieve this, three naive, immunoglobulin gene libraries (a VH domain, a scFv and a Fab library) were interrogated for binders to recombinant NS1 antigen from all four DENV serotypes using phage display technology and various biopanning approaches. From biopanning experiments, four antibody fragments specific to NS1 from each DENV serotype as well as a panel of pan-reactive antibody fragments were isolated. The isolated antibody fragments were reformatted into fully assembled IgG1 antibodies for characterisation. In a sandwich ELISA, none of the pan-reactive antibodies were able to bind DENV NS1 in tandem with the serotype-specific antibodies. All four of the serotype-specific antibodies however, recognised their serotype-respective recombinant and native DENV NS1. The serotype-specific immunoglobulins showed equilibrium dissociation constants that ranged from µM to nM. Used as capture reagents in a sandwich ELISA with a commercial pan-reactive antibody as the detection probe, three of the antibodies (9H2 anti DENV-1 NS1, 7G11 anti DENV-3 NS1 and 6A5 anti DENV-4 NS1) were able to achieve clinically relevant limits of DENV NS1 detection. 4C11, an antibody against DENV-2 NS1 had the poorest performance and was subjected to affinity maturation through targeted mutagenesis of VH CDR3. A new variant A4, showed marginal 2-fold improvement in performance when incorporated into a limit of detection ELISA with the commercial pan-reactive antibody. Our data suggests that the four serotype-specific antibodies, including the new variant, would be useful for inclusion in a sandwich ELISA that uses NS1 to serotype DENV infections. To develop a complete DENV NS1 serotyping assay however, the serotype-specific antibodies will need to be used in a new iteration of a biopanning campaign to isolate an appropriate pan-reactive pairing partner