Detection of Human Adenovirus and Rotavirus in Wastewater in Lusaka, Zambia: Demonstrating the Utility of Environmental Surveillance for the Community.

Enteric infections due to viral pathogens are a major public health concern. Detecting the risk areas requires a strong surveillance system for pathogenic viruses in sources such as wastewater. Towards building an environmental surveillance system in Zambia, we aimed to identify group A rotavirus (RVA) and human adenovirus (HAdV) in wastewater. Convenient sampling was conducted at four study sites every Tuesday for five consecutive weeks. The research team focused on three different methods of viral concentration to determine the suitability in terms of cost and applicability for a regular surveillance system: the bag-mediated filtration system (BMFS), polyethylene glycol-based (PEG) precipitation, and skimmed milk (SM) flocculation. We screened 20 wastewater samples for HAdV and RVA using quantitative polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR). Of the 20 samples tested using qPCR, 18/20 (90%) tested positive for HAdV and 14/20 (70%) tested positive for RVA. For the genetic sequencing, qPCR positives were subjected to cPCR, of which 12 positives were successfully amplified. The human adenovirus was identified with a nucleotide identity range of 98.48% to 99.53% compared with the reference genome from GenBank. The BMFS and SM flocculation were the most consistent viral concentration methods for HAdV and RVA, respectively. A statistical analysis of the positives showed that viral positivity differed by site (p < 0.001). SM and PEG may be the most appropriate options in resource-limited settings such as Zambia due to the lower costs associated with these concentration methods. The demonstration of HAdV and RVA detection in wastewater suggests the presence of the pathogens in the communities under study and the need to establish a routine wastewater surveillance system for the identification of pathogens.

Phylogenetic Analysis of Newcastle Disease Virus Isolated from Poultry in Live Bird Markets and Wild Waterfowl in Zambia.

Poultry production is essential to the economy and livelihood of many rural Zambian households. However, the industry is threatened by infectious diseases, particularly Newcastle disease virus (NDV) infection. Therefore, this study employed next-generation sequencing to characterise six NDV isolates from poultry in Zambia's live bird markets (LBMs) and wild waterfowl. Four NDV isolates were detected from 410 faecal samples collected from chickens in LBMs in Lusaka and two from 2851 wild birds from Lochinvar National Park. Phylogenetic analysis revealed that the four NDVs from LBM clustered in genotype VII and sub-genotype VII.2 were closely related to viruses previously isolated in Zambia and other Southern African countries, suggesting possible local and regional transboundary circulation of the virus. In contrast, the two isolates from wild birds belonged to class I viruses, genotype 1, and were closely related to isolates from Europe and Asia, suggesting the possible introduction of these viruses from Eurasia, likely through wild bird migration. The fusion gene cleavage site motif for all LBM-associated isolates was 112RRQKR|F117, indicating that the viruses are virulent, while the isolates from wild waterfowl had the typical 112ERQER|L117 avirulent motif. This study demonstrates the circulation of virulent NDV strains in LBMs and has, for the first time, characterised NDV from wild birds in Zambia. The study further provides the first whole genomes of NDV sub-genotype VII.2 and genotype 1 from Zambia and stresses the importance of surveillance and molecular analysis for monitoring the circulation of NDV genotypes and viral evolution.

Antimicrobial resistance profiles of Escherichia coli isolated from laying hens in Zambia: implications and significance on one health.

Background: Antimicrobial resistance (AMR) has been deepening in the layer poultry sector in Zambia partly due to the inappropriate use of antimicrobials. Escherichia coli (E. coli), a commensal and zoonotic bacterium, can potentially be a source of AMR. Objectives: This study assessed the phenotypic AMR profiles of E. coli isolated from the apparent health-laying hens in Lusaka and Copperbelt provinces of Zambia. Methods: A cross-sectional study was conducted between September 2020 and April 2021 in which 365 cloacal swabs were collected from 77-layer farms based in Lusaka and Copperbelt provinces of Zambia. E. coli isolation and identification were done using cultural and biochemical properties and confirmed using the 16S rRNA gene sequencing. Antimicrobial susceptibility testing (AST) was done using the Kirby-Bauer disc-diffusion method. Data analysis was done using WHONET 2020 and Stata v.16.1. Results: Of the 365 samples, E. coli was isolated from 92.9% (n = 339). The AMR was detected in 96.5% (n = 327) of the isolates, of which 64.6% (n = 219) were multidrug-resistant (MDR). E. coli was highly resistant to tetracycline (54.6%) and ampicillin (54%) but showed low resistance to meropenem (0.9%), ceftazidime (6.2%) and chloramphenicol (8.8%). Conclusion: This study found a high prevalence of E. coli resistant to some commonly used antibiotics in poultry, which is a public health concern because of the potential contamination of eggs and layers of chicken meat that enter the food chain. Urgent attention is needed, including strengthening antimicrobial stewardship and surveillance programmes in layer poultry production in Zambia.

Luna Virus and Helminths in Wild Mastomys natalensis in Two Contrasting Habitats in Zambia: Risk Factors and Evidence of Virus Dissemination in Semen.

Transmission dynamics and the maintenance of mammarenaviruses in nature are poorly understood. Using metagenomic next-generation sequencing (mNGS) and RT-PCR, we investigated the presence of mammarenaviruses and co-infecting helminths in various tissues of 182 Mastomys natalensis rodents and 68 other small mammals in riverine and non-riverine habitats in Zambia. The Luna virus (LUAV) genome was the only mammarenavirus detected (7.7%; 14/182) from M. natalensis. Only one rodent from the non-riverine habitat was positive, while all six foetuses from one pregnant rodent carried LUAV. LUAV-specific mNGS reads were 24-fold higher in semen than in other tissues from males. Phylogenetically, the viruses were closely related to each other within the LUAV clade. Helminth infections were found in 11.5% (21/182) of M. natalensis. LUAV-helminth co-infections were observed in 50% (7/14) of virus-positive rodents. Juvenility (OR = 9.4; p = 0.018; 95% CI: 1.47-59.84), nematodes (OR = 15.5; p = 0.001; 95% CI: 3.11-76.70), cestodes (OR = 10.8; p = 0.025; 95% CI: 1.35-86.77), and being male (OR = 4.6; p = 0.036; 95% CI: 1.10-18.90) were associated with increased odds of LUAV RNA detection. The role of possible sexual and/or congenital transmission in the epidemiology of LUAV infections in rodents requires further study, along with the implications of possible helminth co-infection.

Isolation, discrimination, and molecular detection of Listeria species from slaughtered cattle in Namwala District, Zambia.

BACKGROUND: The food industry is increasingly becoming more scrutinized, given the frequency and intensity with which zoonotic diseases are being reported. Pathogen tracking has become more applicable with regards food safety. It is in this regard that the present study was formulated to track Listeria species. in freshly slaughtered cattle carcasses by utilizing standard and molecular biological techniques. METHODS: A cross-sectional study design was conducted from March to December 2020 with 200 samples being equally collected in the rainy and dry seasons. A total of 180 and 20 swabs were aseptically collected from carcasses and the environment respectively. Samples were first subjected to pre-enrichment in half-strength Fraser broth followed by enrichment in full strength Fraser broth and subsequent plating on Listeria agar. Listeria growth characteristics were identified up to species level based on their morphological and biochemical characteristics. Further, molecular detection and phylogenetic analysis was conducted. Quantitative proportionate survey data were analyzed using Stata Version 15 software to estimate crude prevalence taking into account complex design at abattoir level. Factors associated with contamination were characterized using logistic regression. Sequences were analyzed using, Genetyyx version 12 and phylogenetic Mega. RESULTS: Of the 200 samples, 19 were positive for Listeria species identified as L.innocua 14/19 (73.7%) and L. monocytogenes 5/19 (26.3%). All isolates were from freshly slaughtered carcasses, and none from environment. Siginificant differences in contamination levels were observed based on season: rainy season yielded 14 (73.6%) whilst the dry season 5 (26.3%). The L. monocytogenes strains showed a high degree of homogeneity on phylogenetic analysis and clustered based on abattoir. Seasonality was identified as a major determinant influencing contamination based on the final logistic regression model. CONCLUSION: This study found evidence of L. monocytogenes contamination on traditionally raised beef carcasses across various abattoirs surveyed. The failure to find Listeria contamination on the abattoir environment may to a greater extent intimate cattle carccases as primary sources of contamination. However, a more comprerehnsive study incorporating different geographical regions is needed to conclusively ascertain these present findings.

Antibiotic Resistance Patterns of Listeria Species Isolated from Broiler Abattoirs in Lusaka, Zambia.

L. monocytogenes is a public health threat linked to fast foods such as broiler chickens. This study aimed to verify the occurrence of Listeria species in chickens from abattoirs and evaluate their antimicrobial resistance. In total, 150 broiler carcass swabs distributed as cloacal (n = 60), exterior surface (n = 60), and environmental (n = 30) were collected. Listeria species were characterized using biochemical tests and PCR. We conducted antibiotic resistance tests using the disc diffusion and Etest (Biomerieux, Durham, NC, USA) methods. Overall isolation of Listeria species was 15% (23/150) 95% CI (10.16-22.33), 2% (3/150) 95% CI (0.52-6.19) and 13% (20/150) 95% CI (8.53-20.08) came from environmental swabs and carcass swabs, respectively. Proportions of positive Listeria isolates were L. monocytogenes 74% (17/23), L. welshimeri 22% (5/23), and L. innocua 4% (1/23). Listeria species from the exterior carcass swabs was 61% (14/23), cloacal swabs 26% (6/23), and environmental swabs 3% (3/23). L. monocytogenes had the greatest resistance percentage to the following antibiotics: clindamycin (61%, 10/23), tetracycline 30% (7/23), and erythromycin 13%, (3/23). Isolation of L. monocytogenes in relatively high numbers, including the antimicrobial profiles, suggests a potential risk of the pathogen remaining viable in the food continuum and a public health risk to would-be consumers.

Identification of Escherichia coli and Related Enterobacteriaceae and Examination of Their Phenotypic Antimicrobial Resistance Patterns: A Pilot Study at A Wildlife-Livestock Interface in Lusaka, Zambia.

A cross-sectional study was used to identify and assess prevalence and phenotypic antimicrobial resistance (AMR) profiles of Escherichia coli and other enterobacteria isolated from healthy wildlife and livestock cohabiting at a 10,000 acres game ranch near Lusaka, Zambia. Purposive sampling was used to select wildlife and livestock based on similarities in behavior, grazing habits and close interactions with humans. Isolates (n = 66) from fecal samples collected between April and August 2018 (n = 84) were examined following modified protocols for bacteria isolation, biochemical identification, molecular detection, phylogenetic analysis, and antimicrobial susceptibility testing by disc diffusion method. Data were analyzed using R software, Genetyx ver.12 and Mega 6. Using Applied Profile Index 20E kit for biochemical identification, polymerase chain reaction assay and sequencing, sixty-six isolates were identified to species level, of which Escherichia coli (72.7%, 48/66), E. fergusonii (1.5%, 1/66), Shigella sonnei (22.7%, 14/66), Sh. flexinerri (1.5%, 1/66) and Enterobacteriaceae bacterium (1.5%, 1/66), and their relationships were illustrated in a phylogenetic tree. Phenotypic antimicrobial resistance or intermediate sensitivity expression to at least one antimicrobial agent was detected in 89.6% of the E. coli, and 73.3% of the Shigella isolates. The E. coli isolates exhibited the highest resistance rates to ampicillin (27%), ceftazidime (14.3%), cefotaxime (9.5%), and kanamycin (9.5%). Multidrug resistance (MDR) was detected in 18.8% of E. coli isolates while only 13.3% Shigella isolates showed MDR. The MDR was detected among isolates from impala and ostrich (wild animals in which no antimicrobial treatment was used), and in isolates from cattle, pigs, and goats (domesticated animals). This study indicates the possible transmission of drug-resistant microorganisms between animals cohabiting at the wildlife-livestock interface. It emphasizes the need for further investigation of the role of wildlife in the development and transmission of AMR, which is an issue of global concern.