Investigation of the governance structure of Nairobi dairy value chain and its influence on food safety

The dairy value chain of Nairobi is comprised, in its majority, of small-scale independent enterprises that operate within a complex interlinked system. In this complexity, the coordination and power structures of the system may have major influences on the management of dairy food safety. Therefore, the aim of this study was to investigate the governance and challenges issues faced by stakeholders throughout the Nairobi dairy value chain and assess their potential implications on food safety. Qualitative data was collected through focus group discussions and key informant interviews based on a dairy value chain mapping framework previously developed. Thematic analysis enabled identification of governance themes, key challenges and their implication on food safety. Themes were organized depending on their association with farmers (informal settlement or peri-urban), dairy cooperatives, dairy traders, processing companies, retailers or government officers. The identified governance themes included: i) weak linkage between government and farmers, ii) inadequate compliance with government regulations by traders and retailers, iii) emphasis on business licenses and permits for revenue rather than for food safety, iv) multiple licensing resulting in high business cost and lack of compliance, v) fragmented regulation, vi) unfair competition and vii) sanctions that do not always result in compliance. The key challenges identified included, amongst others: i) inadequate farmer support, ii) harassment of traders and retailers and iii) high business costs by traders, retailers, dairy cooperatives and large processors. The implication of governance and challenges of food safety were, amongst others: i) inadequate extension services, ii) insufficient cold chain, iii) delivery of adulterated and low milk quality to bulking centres, iv) inadequate food safety training and v) lack of policies for management of waste milk. The range of issues highlighted are based on stakeholders’ perceptions and reflects the complexity of the relationships between them. Many of the governance themes demonstrate the linkages that are both beneficial or confrontational between the formal and informal sectors, and between industry and regulatory authorities, with possible direct food safety consequences. Findings obtained provide indications to decision-makers of potential governance areas that could help improve efficiency and food safety along the dairy value chain

Antimicrobial Usage, Susceptibility Profiles, and Resistance Genes in Campylobacter Isolated from Cattle, Chicken, and Water Samples in Kajiado County, Kenya

Campylobacter organisms are the major cause of bacterial gastroenteritis and diarrhoeal illness in man and livestock. Campylobacter is growingly becoming resistant to critically crucial antibiotics; thereby presenting public health challenge. This study aimed at establishing antimicrobial use, susceptibility profiles, and resistance genes in Campylobacter isolates recovered from chicken, cattle, and cattle-trough water samples. The study was conducted between October 2020 and May 2022 and involved the revival of cryopreserved Campylobacter isolates confirmed by PCR from a previous prevalence study in Kajiado County, Kenya. Data on antimicrobial use and animal health-seeking behaviour among livestock owners (from the same farms where sampling was done for the prevalence study) were collected through interview using a pretested semistructured questionnaire. One hundred and three isolates (29 C. coli (16 cattle isolates, 9 chicken isolates, and 4 water isolates) and 74 C. jejuni (38 cattle isolates, 30 chicken isolates, and 6 water isolates)) were assayed for phenotypic antibiotic susceptibility profile using the Kirby–Bauer disk diffusion method for ampicillin (AX), tetracycline (TE), gentamicin (GEN), erythromycin (E), ciprofloxacin (CIP), and nalidixic acid (NA). Furthermore, detection of genes conferring resistance to tetracyclines (tet (O), β-lactams (blaOXA-61), aminoglycosides (aph-3-1), (fluoro)quinolones (gyrA), and multidrug efflux pump (cmeB) encoding resistance to multiple antibiotics was detected by mPCR and confirmed by DNA sequencing. The correlation between antibiotic use and resistance phenotypes was determined using the Pearson’s correlation coefficient (r) method. Tetracyclines, aminoglycosides, and β-lactam-based antibiotics were the most commonly used antimicrobials; with most farms generally reported using antimicrobials in chicken production systems than in cattle. The highest resistance amongst isolates was recorded in ampicillin (100%), followed by tetracycline (97.1%), erythromycin (75.7%), and ciprofloxacin (63.1%). Multidrug resistance (MDR) profile was observed in 99 of 103 (96.1%) isolates; with all the Campylobacter coli isolates displaying MDR. All chicken isolates (39/39, 100%) exhibited multidrug resistance. The AX-TE-E-CIP was the most common MDR pattern at 29.1%. The antibiotic resistance genes were detected as follows: tet (O), gyrA, cmeB, blaOXA-61, and aph-3-1 genes were detected at 93.2%, 61.2%, 54.4%, 36.9%, and 22.3% of all Campylobacter isolates, respectively. The highest correlations were found between tet (O) and tetracycline-resistant phenotypes for C. coli (96.4%) and C. jejuni (95.8%). A moderate level of concordance was observed between the Kirby–Bauer disk diffusion method (phenotypic assay) and PCR (genotypic assay) for tetracycline in both C. coli (kappa coefficient = 0.65) and C. jejuni (kappa coefficient = 0.55). The study discloses relatively high resistance profiles and multidrug resistance to antibiotics of critical importance in humans. The evolution of the multidrug-resistantCampylobacter isolates has been linked to the use and misuse of antimicrobials. This poses a potential hazard to public and animal health, necessitating need to reduce the use of antibiotics in livestock husbandry practice coupled with stringent biosecurity measures to mitigate antimicrobial resistance

Phenotypic and Genotypic Characterization of Extended Spectrum Beta-Lactamase-Producing Clinical Isolates of Escherichia coli and Klebsiella pneumoniae in Two Kenyan Facilities: A National Referral and a Level Five Hospital

Background. The emergence of antimicrobial resistance (AMR) and multidrug resistance (MDR) among Escherichia coli and Klebsiella pneumoniae, especially through the production of extended spectrum β-lactamases (ESBLs), limits therapeutic options and poses a significant public health threat. Objective. The aim of this study was to assess the phenotypic and genetic determinants of antimicrobial resistance of ESBL-producing Escherichia coli and Klebsiella pneumoniae isolates from patient samples in two Kenyan Hospitals. Methods. We collected 138 E. coli and 127 K. pneumoniae isolates from various clinical specimens at the two health facilities from January 2020 to February 2021. The isolates’ ESBL production and antibiotic susceptibility were phenotypically confirmed using a standard procedure. Molecular analysis was done through conventional polymerase chain reaction (PCR) with appropriate primers for gadA, rpoB, blaTEM, blaSHV, blaOXA, blaCTX-M-group-1, blaCTX-M-group-2, blaCTX-M-group-9, and blaCTX-M-group-8/25 genes, sequencing and BLASTn analysis. Results. Most E. coli (82.6%) and K. pneumoniae (92.9%) isolates were ESBL producers, with the highest resistance was against ceftriaxone (69.6% among E. coli and 91.3% among K. pneumoniae) and amoxicillin/clavulanic acid (70.9% among K. pneumoniae). The frequency of MDR was 39.9% among E. coli and 13.4% among K. pneumoniae isolates. The commonest MDR phenotypes among the E. coli isolates were CRO-FEP-AZM-LVX and CRO-AZM-LVX, while the FOX-CRO-AMC-MI-TGC-FM, FOX-CRO-FEP-AMC-TZP-AZM-LVX-MI and CRO-AMC-TZP-AZM-MI were the most frequent among K. pneumoniae isolates. Notably, the FOX-CRO-FEP-AMC-TZP-AZM-LVX-MI phenotype was observed in ESBL-positive and ESBL-negative K. pneumoniae isolates. The most frequent ESBL genes were blaTEM (42%), blaSHV (40.6%), and blaOXA (36.2%) among E. coli, and blaTEM (89%), blaSHV (82.7%), blaOXA (76.4%), and blaCTX-M-group-1 (72.5%) were most frequent ESBL genes among K. pneumoniae isolates. The blaSHV and blaOXA and blaTEM genotypes were predominantly associated with FOX-CRO-FEP-MEM and CRO-FEP multidrug resistance (MDR) and CRO antimicrobial resistance (AMR) phenotypes, among E. coli isolates from Embu Level V (16.7%) and Kenyatta National Hospital (7.0%), respectively. Conclusions. The high proportion of ESBL-producing E. coli and K. pneumoniae isolates increases the utilization of last-resort antibiotics, jeopardizing antimicrobial chemotherapy. Furthermore, the antimicrobial resistance patterns exhibited towards extended-spectrum cephalosporins, beta-lactam/beta-lactamase inhibitor combinations, fluoroquinolones, and macrolides show the risk of co-resistance associated with ESBL-producing isolates responsible for MDR. Hence, there is a need for regular surveillance and implementation of infection prevention and control strategies and antimicrobial stewardship programs

Cloning, Expression, and Characterization of Babesia gibsoni Dihydrofolate Reductase-Thymidylate Synthase: Inhibitory Effect of Antifolates on Its Catalytic Activity and Parasite Proliferation▿

Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-validated antifolate drug target in certain pathogenic apicomplexans, but not in the genus Babesia, including Babesia gibsoni. Therefore, we isolated, cloned, and expressed the wild-type B. gibsoni dhfr-ts gene in Escherichia coli and evaluated the inhibitory effect of antifolates on its enzyme activity, as well as on in vitro parasite growth. The full-length gene consists of a 1,548-bp open reading frame encoding a 58.8-kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. Each domain contained active-site amino acid residues responsible for the enzymatic activity. The expressed soluble recombinant DHFR-TS protein was approximately 57 kDa after glutathione S-transferase (GST) cleavage, similar to an approximately 58-kDa native enzyme identified from the parasite merozoite. The non-GST fusion recombinant DHFR enzyme revealed Km values of 4.70 ± 0.059 (mean ± standard error of the mean) and 9.75 ± 1.64 μM for dihydrofolic acid (DHF) and NADPH, respectively. Methotrexate was a more-potent inhibitor of the enzymatic activity (50% inhibition concentration [IC50] = 68.6 ± 5.20 nM) than pyrimethamine (IC50 = 55.0 ± 2.08 μM) and trimethoprim (IC50 = 50 ± 12.5 μM). Moreover, the antifolates' inhibitory effects on DHFR enzyme activity paralleled their inhibition of the parasite growth in vitro, indicating that the B. gibsoni DHFR could be a model for studying antifolate compounds as potential drug candidates. Therefore, the B. gibsoni DHFR-TS is a molecular antifolate drug target

Molecular detection and genetic characterisation of pathogenic Theileria, Anaplasma and Ehrlichia species among apparently healthy sheep in central and western Kenya

Tick-borne diseases (TBDs) caused by Theileria, Babesia, Anaplasma and Ehrlichia species are common in tropical and subtropical regions. In this study, we investigated the presence and genetic diversity of Theileria spp., Anaplasma ovis, B. ovis, E. ruminantium and Anaplasma spp. in sheep from the Machakos and Homa Bay counties of Kenya. In order to improve the diagnosis and control of ovine TBDs, a total of 76 blood samples from apparently healthy sheep were screened using a polymerase chain reaction (PCR). The assays were conducted using primers based on Theileria spp. 18S rRNA, Anaplasma ovis Major surface protein-4 (AoMSP4), B. ovis 18S rRNA, E. ruminantium pCS20 and Anaplasma spp. 16S rRNA. The overall infection rates for Theileria spp., A. ovis, E. ruminantium and Anaplasma spp. were 39/76 (51.3%), 26/76 (34.2%), 6/76 (7.9%) and 31/76 (40.8%), respectively. The overall co-infection was 47/76 (61.8%). All Theileria spp. positive samples were confirmed to be of Theileria ovis on sequencing. A phylogenetic analysis of the 18S rRNA gene sequences of T. ovis revealed that all isolates of this study clustered with T. ovis sequences extracted from the GenBank suggesting this gene is highly conserved. E. ruminantium pCS20 sequences were in the same clade on the phylogenetic tree. However, three AoMSP4 sequences from this study appeared in the same clade, while one sequence formed a separate branch revealing genetic divergence. The 16S rRNA sequencing revealed uncharacterised Anaplasma spp. and A. ovis. The phylogenetic analyses of the uncharacterised Anaplasma spp. revealed that the two sequences from this study appear in an independent clade from other sequences extracted from the GenBank. This study provides important information regarding the occurrence of tick-borne pathogens and their degree of genetic diversity among sheep in Kenya, which is useful for the diagnosis and control of TBDs

Global research alliance in infectious disease: a collaborative effort to combat infectious diseases through dissemination of portable sequencing

Objective: To disseminate the portable sequencer MinION in developing countries for the main purpose of battling infectious diseases, we found a consortium called Global Research Alliance in Infectious Diseases (GRAID). By holding and inviting researchers both from developed and developing countries, we aim to train the participants with MinION’s operations and foster a collaboration in infectious diseases researches. As a real-life example in which resources are limited, we describe here a result from a training course, a metagenomics analysis from two blood samples collected from a routine cattle surveillance in Kulan Progo District, Yogyakarta Province, Indonesia in 2019. Results: One of the samples was successfully sequenced with enough sequencing yield for further analysis. After depleting the reads mapped to host DNA, the remaining reads were shown to map to Theileria orientalis using BLAST and OneCodex. Although the reads were also mapped to Clostridium botulinum, those were found to be artifacts derived from the cow genome. An effort to construct a consensus sequence was successful using a reference-based approach with Pomoxis. Hence, we concluded that the asymptomatic cow might be infected with T. orientalis and showed the usefulness of sequencing technology, specifically the MinION platform, in a developing country