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Staphylococcus aureus (SA) food poisoning results from consumption of preformed S. aureus enterotoxins in food. The enterotoxins are one of the most important virulence factors of the bacterium. The risk posed by contamination of milk intended for human consumption by pathogenic S. aureus in pastoral areas in Kenya is still generally not well documented yet this information is critical for ensuring safety to consumers who sometimes may take unpasteurized milk. This study, therefore determined the prevalence of S. aureus enterotoxin genes in raw milk from cattle, goats, sheep and camels intended for human consumption in northern Kenya. A total of 603 milk samples from 57 zebu cattle, 346 galla goats, 8 red Maasai and dorper sheep, 4 one-humped camel (Camelus dromedaries) and 188 pooled from all animals were collected from Isiolo and Marsabit counties of Kenya. S. aureus isolates were cultured from milk samples using a selective media, mannitol salt agar (MSA). Suspect colonies of SA were further analyzed using biochemical tests. Polymerase chain reaction and sequencing techniques were used to confirm SA and detect sea, seb, sec, sed and see enterotoxin genes. Overall, potentially pathogenic S. aureus harboring enterotoxic genes were detected in 85 (14.09%, 95% CI: 11.55–17.1%) of the total milk samples. Genes encoding enterotoxins were detected in the S. aureus bacteria isolated from the milk samples. At least one type of S. aureus enterotoxin gene (SE) was detected in 74.11% (95% CI: 63.91–82.24%) of the 85 isolates. The most frequently encountered gene in the two counties was see (51; 60%, 95% CI: 49.73–69.76%) followed by sea (22; 25.88%, 95% CI: 17.76–36.09%) and sec (19; 22.35%, 95% CI: 14.8–32.29%). None of the isolates tested positive for sed. Overall, 21 of the 85 (24.7%, 95% CI: 16.76–34.83%) strains harbored more than one enterotoxin gene. More than half of the S. aureus isolates harbored at least one of the enterotoxin coding genes, indicating milk samples contaminated by S. aureus could have a high chance of causing staphylococcal food intoxication. Consumption of raw and sour milk in the region could increase the risk of staphylococcal food poisoning and pastoral communities in the region are therefore advised to consume pasteurized milk. • Staphylococcus aureus was detected in 14% of milk samples. • Most of S. aureus (74%) harbored genes that encode for Staphylococcal enterotoxins. • Consumption of raw milk increases risk of staphylococcal food borne disease outbreaks. [ABSTRACT FROM AUTHOR]

Antimicrobial agents are used widely in veterinary and human medicine worldwide. However, their use has suffered a major setback globally due to the emergence of antimicrobial resistance (AMR) in a wide range of microorganisms. This study aimed to investigate the prevalence of AMR in household milk for human consumption in Isiolo County, Kenya. The study identified 42 Escherichia coli (E.coli) isolates from 304 milk samples that were collected in randomly selected households in northern Kenya. E. coli was isolated using Eosin Methylene Blue Agar and identified using biochemical tests. The isolates were confirmed using Polymerase Chain Reaction (PCR) and sequencing. The antimicrobial resistance profiles of the isolates to 11 antimicrobial agents were evaluated by disc diffusion method on Mueller Hinton Agar. Additionally, the isolates were evaluated for antimicrobial genetic determinants conferring the resistance phenotypes to beta-lactams and tetracycline. Overall, 95% of the isolates were resistant to at least one of the tested antimicrobials. Large proportions (81%) of the isolates were resistant to beta-lactams followed by tetracycline (55%) and streptomycin (29%). All the isolates were, however, susceptible to ciprofloxacin and nalidixic acid. Multidrug resistance (MDR) was observed in 14.28% of the isolates and beta-lactam resistant isolates were confirmed to be harbouring bla SHV , bla TEM and bla CTX-M genes. The demonstration of AMR determinants and especially Extended Spectrum Beta Lactamase (ESBL) carriers in milk reveals the risk posed to food safety, particularly to communities that consume raw milk. This study found that raw milk consumed in Isiolo County was contaminated with resistant strains of E. coli. There is, therefore, a need to determine the sources of this resistance and implement interventions to reduce the emergence and spread of AMR bacteria. We recommend implementation of measures that could reduce the presence of antimicrobial resistant E. coli strains in raw milk food chain. • E. coli was detected in 13.8% of the milk samples. • Ninety five (95%) of E. coli were resistant to at least one of the tested antimicrobials. • E. coli was highly resistant to beta-lactams (81%) and tetracycline (55%). • Multidrug resistance (MDR) was observed in 14.28% of the E. coli isolates. [ABSTRACT FROM AUTHOR]

Abstract: The inosine monophosphate dehydrogenase (IMPDH) enzyme has been characterized and validated as a molecular drug target in other apicomplexans but not in the genus Babesia. Subsequently, we cloned and expressed a Babesia gibsoni IMPDH (BgIMPDH) cDNA in Escherichia coli. We also determined the inhibitory effect of mycophenolic acid (MPA) on recombinant BgIMPDH (rBgIMPDH) activity and the Babesia-growths in vitro. The translated BgIMPDH peptide contained thirteen amino acid residues responsible for substrate and cofactor binding in its catalytic domain with Gly374 in BgIMPDH being replaced by Ser388 in mammalian IMPDH. The native BgIMPDH enzyme in the parasite was approximately 54-kDa a mass similar to His-tag rBgIMPDH protein. The K m values of the rBgIMPDH were 8.18±0.878 (mean±standard error of the mean) μM and 360.80±43.41μM for IMP and NAD+, respectively. MPA inhibited the rBgIMPDH activity yielding a K i value of 20.93±1.83μM with respect to NAD+. For Babesia growths, the IC50s were 0.95±0.21 and 2.88±0.49μM for B. gibsoni and B. bovis, respectively. Therefore, our results suggest that MPA may inhibit the replication of Babesia parasites by targeting IMPDH enzyme of the purine pathway. [Copyright &y& Elsevier]

Abstract: We determined the molecular characteristics of four proteins, BgP32, BgP45, BgP47, and BgP50, of Babesia gibsoni. Localization by subcellular fractionations followed by Western blotting revealed that the corresponding native proteins belong to merozoite surface protein family of B. gibsoni (BgMSP). Moreover, antisera against either rBgP45 or rBgP47 cross-reacted with all the proteins of the BgMSP family on ELISA and IFAT analyses. Of the four candidate antigens, ELISA with rBgP45 yielded high sensitivity, and ELISA with rBgP32 resulted in high specificity and in concordance with IFAT results. [Copyright &y& Elsevier]

Abstract Demand for dairy products in sub-Saharan Africa, is expected to triple by 2050, while limited increase in supply is predicted. This poses significant food security risk to low income households. Understanding how the dairy food system operates is essential to identify mitigation measures to food insecurity impact. This study aims to determine the structure and functionality of Nairobi's dairy system using a value chain mapping approach. Primary data were gathered through focus group discussions and key informant interviews with dairy value chain stakeholders in Nairobi to obtain qualitative information on people and products in the chains while describing their interactions and flows. Qualitative thematic analysis combined with flowcharts created by participants enabled identification of key food system segments and the development of chain profiles (or flow-diagrams) which together form Nairobi's dairy system. Seven chain profiles forming Nairobi's dairy value chain were identified. These were found to be dominated by small-scale individuals who operate largely independently. Our profiles for the urban and peri-urban farming systems were structurally similar in their downstream networks, obtaining inputs from similar sources. Upstream, the urban systems were shorter, supplying mostly to immediate neighbours or based on own consumption, while the peri urban systems supplied to a wider network and showed some affiliations to producers' associations. Two distinct profiles characterize the milk flow from traders belonging either to a Dairy Traders Association (DTA) or those not belonging to this association (non-DTA). DTA traders sell mainly to fixed retailers and non-DTA traders to mobile retailers (hawkers or roadside vendors). Profiles associated with medium and large cooperatives were driven by networks of collection centres, but with medium-sized cooperatives selling half of their production to large processing companies, and large cooperatives only to fixed retailers. Large processing companies' profiles indicated distribution of high volumes and value addition processing. They reported strategic milk collection arrangements with suppliers on long, medium - or short - term contracts and with well-established product distribution channels. We have identified numerous inter-linkages across dairy chain profiles in Nairobi's complex system, demonstrating significant interdependency among the stakeholders. Therefore, enhancing the system's efficiency requires a holistic, system-wide approach and any policy interventions should consider every segment of the value chain. This study provides a methodological approach for organizations and policy makers to understand and address structural and functional vulnerabilities within food systems more broadly. The insights from this study are relevant to other rapidly growing cities in the region. Highlights • The structure of Nairobi's complex dairy value chain is provided. • Inter-dependency and inter-linkages of the stakeholders involved in the dairy chain is described • To enhance the system's efficiency, interventions require a holistic approach targeting every segment of the value chain [ABSTRACT FROM AUTHOR]

Abstract: Toxoplasma gondii is an intracellular protozoan parasite, which relies on a specialized compartment, the parasitophorous vacuole (PV), to survive within host cells. Dense granules within the parasite release a large variety of proteins to maintain the integrity of the vacuole structure. Here, we identified a novel dense granule protein in T. gondii, TgGRA23, which is a homolog of the Sarcocystis muris dense granule protein, SmDG32. Recombinant TgGRA23 (rTgGRA23) expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein was used to raise antisera in mice and rabbits. Immunoblotting showed that antisera from the immunized mice and rabbits reacted with parasite lysates to yield a 21-kDa native protein. In addition, immuno-electron microscopic examination showed that TgGRA23 resides in the dense granules, PV membrane and intravacuolar network of the parasite. To confirm the precise subcellular localization of TgGRA23 in T. gondii, an immunofluorescent antibody test was performed using dense granule markers. Notably, TgGRA23 co-localized with other dense granule proteins including TgGRA4 and TgGRA7, in the extracellular-stage parasites. Biochemical experiments indicated that TgGRA23 is insoluble and may form an electrostatic complex that is resistant to non-ionic detergents. Furthermore, specific antibodies to TgGRA23 were detected during the chronic stage of Toxoplasma infection in mice. Our results suggest that TgGRA23 is an as yet unknown member of the T. gondii dense granule proteins, and that it may be involved in remodeling or maintenance of the PV. [Copyright &y& Elsevier]

Abstract: A cDNA encoding farnesyl pyrophosphate synthase of Babesia bovis (BbFPPS) has been isolated, cloned and characterized as molecular drug target. Sequence analysis revealed that BbFPPS contains an open reading frame of 1011bp with predicted 336 amino acids and molecular mass of 38kDa. Antiserum raised in mice against recombinant BbFPPS expressed in Escherichia coli specifically reacted with native protein of B. bovis parasites by Western blot analysis and indirect immunofluorescent test. Enzymatic assay using recombinant BbFPPS revealed that the Km value of the enzyme for isopentenyl pyrophosphate and dimethylallyl pyrophosphate was 2.494±1.536μM. Risedronate inhibited the activity of BbFPPS yielding IC50 value of 8.4±1.2nM. Furthermore, the in vitro growth of B. bovis was significantly inhibited in the presence of a micromolar concentration of risedronate (IC50 =4.02±0.91μM). No regrowth of B. bovis was observed at 10μM of risedronate in the subsequent viability test. These results demonstrate that BbFPPS is the molecular target of risedronate, which could inhibit the in vitro growth of B. bovis. [Copyright &y& Elsevier]

Abstract: A heterologous prime-boost strategy with priming plasmid DNA followed by recombinant virus expressing relevant antigens is known to stimulate protective immunity against intracellular parasites. In this study, we have evaluated a heterologous prime-boost strategy for immunizing mice against Toxoplasma gondii infection. Our results revealed that the prime-boost strategy using both plasmid DNA and adenoviral vector encoding TgAMA1 may stimulate both humoral and Th1/Th2 cellular immune responses specific for TgAMA1. Moreover, C57BL/6 mice immunized with the pAMA1/Ad5Null, pNull/Ad5AMA1, and pAMA1/Ad5AMA1 constructs showed survival rates of 12.5%, 37.5%, and 50%, respectively. In contrast, all the pNull/Ad5Null immunized mice died after infection with the PLK-GFP strain of T. gondii. Brain cyst burden was reduced by 23% in mice immunized with pAMA1/Ad5AMA1 compared with the pNull/Ad5AMA1 immunized mice. These results demonstrate that the heterologous DNA priming and recombinant adenovirus boost strategy may provide protective immunity against T. gondii infection. [Copyright &y& Elsevier]