The 2023 anthrax outbreak in Zambia, spanning from May to November, resulted in 390 livestock and 39 wildlife fatalities. In addition, 834 suspected human cases and 4 fatalities were recorded. This paper reports the genome sequences of the Bacillus anthracis isolated from humans and soil during the outbreak.

Ticks harbor diverse Rickettsia spp., including several spotted fever group rickettsiae of veterinary and public health importance. However, information on tick-associated Rickettsia spp. in Zambia remains geographically limited. In this retrospective molecular survey, we examined 1,587 individual ticks collected from vegetation and domestic animals in eight provinces of Zambia during 2016-2018. Ticks were identified morphologically and screened for Rickettsia spp. by PCR targeting the citrate synthase gene (gltA). Representative gltA positive samples were further characterized by sequencing of ompA, ompB, htrA, sca4, and the 16S rRNA gene. In total, 204 ticks (12.9%) were tested positive for gltA and detected in all eight provinces examined. Sequencing of gltA revealed 26 sequence variants, of which 25 clustered within the spotted fever group and one belonged to transitional group rickettsiae. Multilocus phylogenetic analysis identified sequence variants related to recognized or putative tick-borne rickettsiae, including R. africae, R. conorii, R. massiliae, R. aeschlimannii, Candidatus R. jingxinensis, Candidatus R. rhabdomydis, and R. hoogstraalii. These findings indicate that ticks in Zambia harbor genetically diverse Rickettsia lineages and provide a geographically broad molecular baseline for future surveillance, isolation attempts, and genomic characterization of tick-borne rickettsiae in the region.

Newcastle disease (ND) remains one of the most devastating infectious diseases of poultry worldwide, causing substantial economic losses despite the widespread use of vaccination programs. In Zambia, recurrent outbreaks continue to be reported. This study aimed to characterise the pathological features and molecular diversity of Newcastle disease virus (NDV) circulating in poultry outbreaks in Lusaka Province, Zambia. A prospective and retrospective investigation of suspected ND outbreaks reported between 2020 and 2023 was conducted. A total of 89 poultry outbreaks involving indigenous chickens, broilers, layers, and pullets were investigated. Gross pathology and histopathological examinations were performed on affected birds, followed by molecular detection of NDV using reverse transcription polymerase chain reaction (RT-PCR). Partial fusion (F) gene sequencing and phylogenetic analysis were used to determine circulating genotypes. Gross lesions were consistent with ND but lacked specificity, while histopathology revealed predominantly necro-haemorrhagic lesions affecting lymphoid tissues, gastrointestinal tract, and respiratory organs, with variable severity across production systems. Newcastle disease virus was detected in both vaccinated and unvaccinated flocks, with no statistically significant difference in detection rates between the two groups (p > 0.05). Molecular analysis identified the circulation of virulent NDV genotype VII.2, alongside genotype II strains closely related to vaccine lineages. The detection of virulent NDV genotypes in vaccinated flocks highlights persistent gaps in Newcastle disease control in Zambia. These findings underscore the need for strengthened molecular surveillance, improved vaccination strategies, and enhanced biosecurity to reduce the burden of ND in poultry production systems.

Background and Aim:Non-typhoidal Salmonella remains a significant zoonotic pathogen with substantial implications for animal and public health, particularly in regions where domestic animals and wildlife share ecological interfaces. The invasion protein A (invA) gene is widely used as a molecular marker for detecting Salmonella; however, its sequence variability across host species in sub-Saharan Africa remains poorly understood. This study aimed to characterize nucleotide diversity and phylogenetic relationships of the invA gene among Salmonella isolates obtained from domestic animals and free-ranging wildlife in selected regions of Zambia. Materials and Methods:A total of 12 invA gene amplicons derived from previously confirmed Salmonella isolates from domestic animals (n = 10) and wildlife (n = 2) were selected for sequencing. Polymerase chain reaction products were purified and sequenced using BigDye terminator chemistry on a 3500 Genetic Analyzer. Sequence assembly and editing were performed using GENETYX software. Multiple sequence alignment was performed with ClustalW, and phylogenetic relationships were inferred using maximum-likelihood with the Tamura-Nei model in MEGA7. Comparative analysis included eight reference sequences retrieved from GenBank. Results:The invA gene sequences demonstrated high nucleotide similarity (98.0%-100.0%) among isolates from domestic animals and wildlife, while showing broader variability (74.0%-100.0%) when compared with global reference strains. Six distinct sequence types were identified, with the majority originating from chicken isolates. Nucleotide substitutions were predominantly observed between positions 40 bp and 250 bp, indicating a potential hotspot for genetic variation. Some substitutions resulted in amino acid changes, suggesting possible structural and functional implications for the invasion protein. Phylogenetic analysis revealed that all Zambian isolates clustered within a single major lineage (Clade A), indicating close genetic relatedness across host species. Conclusion:This study provides the first evidence of invA gene sequence polymorphism among Salmonella isolates from domestic animals and wildlife in Zambia. The findings highlight the dynamic evolution of Salmonella at the wildlife-livestock interface and underscore the importance of molecular surveillance in understanding zoonotic transmission. The identified sequence variations may have implications for diagnostic accuracy and the development of region-specific detection tools, reinforcing the need for continuous genomic monitoring under a One Health framework.

In Zambia, 40% of clinical Gram-negative bacteria are either Escherichia coli or Klebsiella pneumoniae, with a high third-generation cephalosporin (3GC) resistance prevalence. Therefore, 3GC resistance surveillance is a crucial indicator for guiding focused intervention policies. However, the lack of genotypic diagnostic tools limits the ability to elucidate trends, especially in peri-urban and rural areas of developing countries. This study aimed to develop a rapid, cost-effective tool for the genotypic surveillance of 3GC resistance. Here, 900 stool samples collected from patients in Kafue (peri-urban, n = 400) and Katete (rural, n = 500) districts of Zambia were used for bacterial isolation on MacConkey agar supplemented with 1 μg/ml cefotaxime. Isolated 3GC-resistant strains were characterized by sequencing the 16S rRNA gene and screening for blaCTX-M and blaTEM genes using single polymerase chain reaction (PCR). Furthermore, selected 3GC-resistant strains were subjected to whole-genome sequencing (WGS) using MiSeq/HiSeq (n = 34) and MinION (n = 1). Using the data from this and other previous studies, we developed a rapid PCR-dipstick DNA chromatography-based tool for detecting blaCTX-M, blaTEM, E. coli-specific yaiO, and K. pneumoniae-specific khe genes. The prevalence of isolated 3GC resistant strains was 15.4% (139/900), dominated by E. coli (102/139, 73.4%). On PCR, the blaCTX-M gene was detected in 72.7% (101/139) of the isolates, while blaTEM was found in 46.8% (65/139) of the strains. The developed tool displayed a high level of agreement with WGS and single PCR/Sanger sequencing, with sensitivity and specificity ≥ 95% and Kappa ≥ 0.95 for each of the four target genes. We envisage that the simplicity and adaptability of this tool will be a significant advantage for the surveillance of 3GC resistance in Zambia and elsewhere.

Bats are widely recognized as reservoirs of emerging and re-emerging pathogens, and their ecological interactions with humans and livestock present important opportunities for the transmission of infectious agents and antimicrobial resistance (AMR). However, little is known about the occurrence of resistant bacteria in bat-associated environments in Zambia or their potential role in the maintenance of AMR outside clinical and agricultural settings. This study investigated the genomic characteristics of cefotaxime-resistant Proteobacteria isolated from bat fecal droppings collected at Leopards Hill Cave, an established hotspot for zoonotic pathogens. Four hundred bat fecal samples were cultured on cefotaxime-supplemented MacConkey agar, and those exhibiting bacterial growth were subjected to antimicrobial susceptibility testing and whole-genome analysis. Of the 400 samples processed, four (1 %) yielded growth, resulting in three bacterial species: Pseudomonas aeruginosa (n = 1), Enterobacter mori (n = 1), and Brucella intermedia (formerly Ochrobactrum intermedium) (n = 2). Genomic screening revealed that P. aeruginosa strain CB_234 harbored bla OXA-50, aph(3')-IIb, and catB7, which confer resistance to β-lactams, aminoglycosides, and chloramphenicol, respectively. It also possessed multiple virulence determinants involved in adherence, motility, and secretion systems that enhance host colonization and environmental persistence. Core genome phylogenetic analysis placed CB_234 within a clade exclusively composed of clinical isolates from Nigeria, Thailand, Russia, Kenya, and Ghana, indicating a shared evolutionary lineage among globally dispersed hospital-associated strains. Conversely, environmental isolates from plant and aquatic sources, along with a dog-associated isolate, were phylogenetically distant, highlighting the distinct evolutionary origins. The E. mori isolate carried bla ACT and qnrE resistance genes and plasmid replicons, suggesting potential mobility of resistance traits through horizontal gene transfer. In contrast, the two B. intermedia isolates did not harbor any known AMR genes or plasmid replicons. However, this species is increasingly recognized as an opportunistic pathogen. The detection of AMR-associated bacterial species in a natural bat habitat supports the evidence of resistance determinants circulating in wildlife environments in Zambia. Given that bats are unlikely to encounter clinical antibiotics directly, the persistence of such genes in their environment suggests that natural ecosystems may play an underappreciated role in maintaining AMR reservoirs independent of direct antimicrobial pressure. These findings underscore the importance of incorporating wildlife and environmental niches into national and global AMR surveillance frameworks under a One Health approach to better understand the ecological dimensions of AMR emergence and dissemination.

Antimicrobial resistance (AMR) mediated by extended-spectrum β-lactamases (ESBLs) is a growing global concern, particularly among Enterobacteriaceae. The CTX-M-type ESBLs, encoded by the blaCTX-M gene, are of significant public health importance due to their high prevalence and broad geographic distribution. Typically located on plasmids and often co-occurring with other AMR genes, blaCTX-M contributes to multidrug resistance (MDR). However, increasing evidence suggests secondary chromosomal integration of blaCTX-M, sometimes alongside other resistance determinants. The extent and implications of this mechanism remain poorly characterized, especially in Africa, where genomic surveillance is limited. In this study, we retrieved 295 chromosomal sequences of Enterobacteriaceae of African origin from the GenBank and performed in silico predictions of blaCTX-M and other AMR genes. blaCTX-M-carrying sequences were further characterized by in silico multilocus sequence typing and genome annotation. Chromosomal insertions were identified through alignment with reference genomes. Overall, 47 of 295 sequences (15.9%) harbored the blaCTX-M gene, with the highest prevalence in Klebsiella pneumoniae (29/157, 18.5%), followed by Escherichia coli (13/72, 18.1%), Enterobacter spp. (4/38, 10.5%), and Shigella spp. (1/12, 8.3%). The most common allele was blaCTX-M-15 (31/47, 66.0%), followed by blaCTX-M-14 (12/47, 25.5%), blaCTX-M-55 (3/47, 6.4%), and blaCTX-M-27 (1/27, 3.7%). Co-occurrence of blaCTX-M with additional AMR genes was frequently observed, with integration events often associated with mobile genetic elements such as ISEcp1 and IS26. Notably, strains from the same hospital setting were phylogenetically related and shared sequence types and AMR gene profiles, suggesting local clonal dissemination. These findings reveal a notable presence of chromosomally integrated blaCTX-M among African Enterobacteriaceae, frequently in association with other resistance genes, thereby facilitating stable MDR propagation independent of plasmid maintenance. This evolutionary adaptation may have significant implications for the persistence and spread of MDR in clinical settings.

Rotavirus C (RVC) causes acute gastroenteritis in neonatal piglets. Despite the clinical importance of RVC infection, the distribution and prevalence in pig populations in most African countries remains unknown. In this study, we identified RVC in Zambian pigs by metagenomic analysis. The full genome sequence of the RVC revealed two different VP4 sequences, implying that two different RVC strains (ZP18-77-c1 and ZP18-77-c2) were present in the same sample. Genetic analyses demonstrated that all segments of ZP18-77-c1 and ZP1877-c2 showed high nucleotide sequence identities (87.7-94.5%) to known porcine RVC strains, and ZP18-77c1 and ZP18-77-c2 strains were assigned to genotype constellations, G1-P[4]/P[14]-I13-R5-C5-M1-A7-N9-T10E5-H1. We further screened RVC genomes among pig feces collected in Zambia (n = 147) by RT-qPCR, and 78 samples (53.1%) were positive. This study demonstrated the first full genome sequence of African RVC strains with a relatively high prevalence of RVC infection in the pig populations in Zambia.

Enteric infections are a major public health concern. Detecting risk areas requires a strong surveillance system for pathogenic viruses in possible sources such as wastewater. To build an environmental surveillance system, we aimed to identify group A rotavirus (RVA) and human adenovirus (HAdV) in wastewater. We also focused on three different methods of viral concentration to determine suitability for a regular surveillance system. We screened 20 wastewater samples for HAdV and RVA using quantitative polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR). Sixteen posi-tives (18/20) and ten (14/20) positives for HAdV and RVA, respectively, were obtained on qPCR, representing a prevalence of 90% and 70%. Twelve (12) positives were successfully amplified. Bag-mediated filtration system (BMFS) and skimmed milk (SM) flocculation were the most consistent viral concentration methods for HAdV and RVA, respectively. Human adenovirus was identified with nucleotide identity range of 98.48% to 99.53%. Statistical analysis of positives showed that viral burden differed by site (P

Urogenital schistosomiasis remains a pervasive health challenge in rural Zambian communities. This study explores the molecular epidemiology and genetic diversity of Schistosoma haematobium using mitochondrial genes (cox1 and nadh1). Urine samples from 421 children in Siavonga and Lusaka districts, Zambia, were collected between December 2020 and February 2022. Microscopy and DNA extraction facilitated the identification of S. haematobium, followed by amplification, sequencing, and phylogenetic analysis of cox1 and nadh1 genes. Phylogenetic analysis revealed clustering with samples from mainland African countries, emphasizing shared haplotypes. Both mitochondrial genes exhibited substantial diversity, with 5 haplotypes from 37 cox1 sequences and 12 haplotypes from 23 nadh1 sequences. High haplotype diversity (0.621-0.808) and low nucleotide diversity (0.00181-0.03288) were observed. Siavonga and Lusaka districts shared the majority of S. haematobium haplotypes. Molecular variance and genetic differentiation analysis indicated variations within populations rather than between populations (cox1: -0.025, nadh1: 0.01646). These findings suggest a limited differentiation between S. haematobium populations in Siavonga and Lusaka, potentially indicating gene flow. Tajima's test revealed negative values, indicating a departure from neutrality, introduction of rare alleles, and recent population expansion. This study contributes essential insights into S. haematobium population genetics, crucial for effective urogenital schistosomiasis control in Zambia.

Background Rodent-borne orthohantaviruses are the causative agents of hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Apart from the classic rodent-borne hantaviruses, numerous species of hantaviruses have been identified in shrews and bats; however, their antigenicity and pathogenicity are unknown. This study focused on developing a serological method to detect antibodies against bat- and shrew-borne hantaviruses. Methodology/Principal findings Five bat-borne (Brno, Dakrong, Quezon, Robina, and Xuan Song) and 6 shrew-borne (Asama, Altai, Cao Bang, Nova, Seewis, and Thottapalayam) viruses were selected based on the phylogenetic differences in their N proteins. The recombinant N (rN) proteins of these viruses were expressed as antigens in Vero E6 and 293T cell lines using the pCAGGS/MCS vector. Antisera against the Nus-tagged rN fusion proteins of these viruses (mouse anti-Brno, Dakrong, Quezon, Robina, Xuan Song, Asama, Cao Bang, and Nova, while rabbit anti-Altai, Seewis and Thottapalayam) were also generated. Antigenic cross-reactivity was examined in antisera and rN-expressing Vero E6 cells. The rN proteins of almost all the tested viruses, except for the Quezon and Robina viruses, showed independent antigenicity. For serological screening of bat samples, 5 rNs of the bat-borne viruses were expressed together in a single transfection protocol. Similarly, 6 rNs of shrew-borne viruses were expressed. Reactivities of the mixed antigen system were also examined across the singly transfected Vero cell lines to ensure that all antigens were expressed. Using these antigens, bat serum samples collected from Zambia were screened using the indirect immunofluorescence antibody test (IFAT). Selected positive samples were individually tested for the respective antigens by IFAT and western blot assays using rN-expressing 293T cell lysates. Of the 1,764 bat serum samples tested, 11.4% and 17.4% were positive for bat and shrew mixed antigens, respectively. These samples showed positive reactions to the Brno, Dakrong, Quezon, Xuan Son, Robina, Asama, Altai, Cao Bang, or Thottapalayam virus antigens. Conclusions/Significance These observations suggest that the mixed-antigen screening system is useful for serological screening For Orthohantavirus infections and that bats in Zambia are likely exposed to not only bat-borne hantaviruses but also to shrew-borne hantaviruses.

West Nile virus (WNV) is a mosquito-borne flavivirus that causes encephalitis in humans and infects crocodiles, resulting in rashes and neurological signs. In Zambia, two distinct lineages of WNV have been detected in neighbouring areas: lineage 2 in mosquitoes and lineage 1a in farmed crocodiles. Considering the risk of direct or vector-mediated WNV transmission from crocodiles to mammals, it is necessary to elucidate the pathogenicity of WNV strains derived from crocodiles. In this study, WNV was successfully isolated from naturally infected farmed crocodiles (Croc110/2019/1/ZM, Croc110). We then investigated its proliferation and pathogenicity in mice in comparison with a WNV isolate from mosquitoes in Zambia (Zmq16) and two reference strains, including one highly pathogenic (NY99) and one low pathogenic (Eg101) strain. Although viral proliferation in Vero and mammalian neuronal cells was comparable among the strains, Croc110 exhibited low cell-to-cell transmission efficiency. In vivo, more than 70% of mice (C57BL/6) intracerebrally inoculated with Croc110 displayed neurological signs, and Croc110-infected mice exhibited similarly high mortality rates as NY99- and Zmq16-infected mice. Meanwhile, comparable virus growth was observed among the strains in the brain. However, the virulence of Croc110 was significantly lower than that of Zmq16 and NY99 following intradermal (ID) and intraperitoneal inoculation. Consistently, Croc110 displayed lower growth than Zmq16 and NY99 in the brain and peripheral tissues after ID inoculation. Our study revealed that the crocodile-derived WNV strain is less neuroinvasive in mice, and it exhibits distinct pathogenicity from the highly pathogenic mosquito-derived WNV strain circulating in Zambia.

Poultry products in Zambia form an integral part of the human diet in many households, as they are cheap and easy to produce. The burden of poultry diseases has, however, remained a major challenge. Growing consumer demand for poultry products in Zambia has resulted in non-prudent antimicrobial use on farms, intending to prevent and treat poultry diseases for growth optimisation and maximising profits. This cross-sectional study aimed to identify the different types of bacteria causing diseases in chickens in Lusaka and to detect the extended-spectrum lactamase (ESBL)-encoding genes. We collected 215 samples from 91 diseased chickens at three post-mortem facilities and screened them for Gram-negative bacteria. Of these samples, 103 tested positive for various clinically relevant Enterobacteriaceae, including Enterobacter (43/103, 41.7%), Escherichia coli (20/103, 19.4%), Salmonella (10/103, 9.7%), and Shigella (8/103, 7.8%). Other isolated bacteria included Yersinia, Morganella, Proteus, and Klebsiella, which accounted for 21.4%. E. coli, Enterobacter, Salmonella, and Shigella were subjected to antimicrobial susceptibility testing. The results revealed that E. coli, Enterobacter, and Shigella were highly resistant to tetracycline, ampicillin, amoxicillin, and trimethoprim-sulfamethoxazole, while Salmonella showed complete susceptibility to all tested antibiotics. The observed resistance patterns correlated with antimicrobial usage estimated from sales data from a large-scale wholesale and retail company. Six (6/14, 42.9%) E. coli isolates tested positive for blaCTX-M, whilst eight (8/14, 57.1%) Enterobacter samples tested positive for blaTEM. Interestingly, four (4/6, 66.7%) of the E. coli isolates carrying blaCTX-M-positive strains were also positive for blaTEM. Sanger sequencing of the PCR products revealed that five (5/6, 83.3%) of the abovementioned isolates possessed the blaCTX-M-15 allele. The results suggest the presence of potentially pathogenic ESBL-producing Enterobacteriaceae in poultry, threatening public health.

Rotavirus B (RVB) causes diarrhea in humans and pigs. Although various RVB strains were identified in humans and various animals globally, little is known about the epidemiology RVB infection in Africa. In this study, we attempted to examine the prevalence of RVB infection in pig populations in Zambia. Metagenomic analyses were conducted on pig feces collected in Zambia to detect double stranded RNA viruses, including RVB. To clarify the prevalence of RVB infection in pig populations in Zambia, 147 fecal samples were screened for the RVB detection by RT-qPCR. Full genome sequence of a detected RVB was determined by Sanger sequencing and genetically analyzed. The metagenomic analyses revealed that RVB sequence reads and contigs of RVB were detected from one fecal sample collected from pigs in Zambia. RT-qPCR screening detected RVB genomes in 36.7

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.

Introduction Trypanosomiasis is caused by several species of trypanosomes. The disease is endemic in Eastern Zambia, particularly in the Luangwa River valley. There is a significant threat as many people are at risk of the infection. The disease is classified as a neglected tropical disease. Previously, Trypanosome epidemiological studies in this area focused on using low- sensitive parasitological diagnostic tools to determine the prevalence in animals and some few investigations on trypanosomiasis infection rates in tsetse flies. Livestock production, crop production and jobs in the tourism sector are the primary sources of livelihood in the south Luangwa national park. The South Luangwa National Park settlement town of Mfuwe is an economically and ecologically important area. The presence of trypanosomiasis repetitively threatens tourism and livelihoods, hence the forthcoming need to clarify the trypanosomiasis situation and to advocate the best tsetse and trypanosomiasis control measures in the area. These threats negatively affect the country’s Forex because fewer tourists are visiting. Material and methods A cross-sectional study that was conducted in Mfuwe of the Eastern Province of Zambia to determine trypanosome infections in tsetse flies. Trapping of the tsetse flies involved the use of epsilon traps and black screen fly rounds in four different vegetation zones, namely open savannah, closed savannah, Montane and riverine. Diagnosis of trypanosome infections in trapped tsetse flies was achieved using a nested PCR which employed the use of two sets of primers targeting the ITS genes. Results Two hundred twenty-three (223) tsetse flies were captured, and only 213 were processed for laboratory analysis. The highest catches from the areas selected were (153) of Tsetse flies in montane, followed by areas near the open forest (lagoons), (48) whereas the lowest catches was recorded in the riverine areas with 22 catches. The species found in these areas were Glossina morsitans morsitans and Glossina palpalis, but Glossina brevipalpis were not found,. The overall prevalence using a nested PCR of the identified trypanosome infection rates in tsetse flies was 18.3% (p < 0.001). The difference in prevalence between sampling areas were significant. Statistically significant differences (p < 0.001) were observed when the prevalence of trypanosome infections was compared by season. The Trypanosoma species found were T.congolense, T.vivax, T.brucei and T.theileri. Conclusions The results of this study showed that location has a significant contribution to the trypanosome infection rates in the tsetse vector at the Wildlife-Livestock-human interface of the Luangwa valley. The results are essential for designing community-wide tsetse and trypanosomiasis control interventions and planning sustainable regimes for mitigating the burden of trypanosomiasis. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The author(s) received no specific funding for this work. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: N/A I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes N/A

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.

We report sequences of the complete linear chromosome and five linear plasmids of the relapsing fever spirochete “Candidatus Borrelia fainii” Qtaro. The chromosome sequence of 951,861 bp and the 243,291 bp of plasmid sequences were predicted to contain 852 and 239 protein-coding genes, respectively. The predicted total GC content was 28.4%.

In recent years, the southern African region has experienced repeated incursions of highly pathogenic avian influenza viruses (HPAIVs), with wild migratory birds being implicated in the spread. To understand the profile of avian influenza viruses (AIVs) circulating in Zambia, we surveyed wild waterfowl for AIVs and phylogenetically characterised the isolates detected in 2015, 2020, and 2021. A total of 2,851 faecal samples of wild waterfowl were collected from Lochinvar National Park in the Southern Province of Zambia. During the study period, 85 (3.0%) low pathogenicity AIVs belonging to various subtypes were isolated, with H2N9, H8N4, and H10N8 being reported for the first time in avian species in Africa. The majority of the isolates were detected from glossy ibis (order Pelecaniformes) making it the first report of AIV from these birds in Zambia. Phylogenetic analysis of all eight gene segments of the 30 full genomes obtained in this study revealed that all the isolates belonged to the Eurasian lineage with their closest relatives being viruses isolated from wild and/or domestic birds in Bangladesh, Belgium, Egypt, Georgia, Mongolia, the Netherlands, and South Africa. Additionally, the Zambian viruses were grouped into distinct clusters based on the year of isolation. While no notifiable AIVs of the H5 or H7 subtypes were detected in wild birds in Zambia, viral internal protein genes of some viruses were closely related to H7 low pathogenicity AIVs. This study shows that periodically, a considerable diversity of AIV subtypes are introduced into the Zambian ecosystem by wild migratory waterfowl. The findings highlight the importance of continuous surveillance and monitoring of AIVs in wild waterfowl, including birds traditionally not considered to be major AIV reservoirs, for a better understanding of the eco-epidemiology and evolutionary dynamics of AIVs in Africa.

Tsetse flies are obligate hematophagous vectors of animal and human African trypanosomosis. They cyclically transmit pathogenic Trypanosoma species. The endosymbiont Sodalis glossinidius is suggested to play a role in facilitating the susceptibility of tsetse flies to trypanosome infections. Therefore, this study was aimed at determining the prevalence of S. glossinidius and trypanosomes circulating in tsetse flies and checking whether an association exists between trypanosomes and Sodalis infections in tsetse flies from Kafue National Park in Zambia. A total of 326 tsetse flies were sampled from the Chunga and Ngoma areas of the national park. After DNA extraction was conducted, the presence of S. glossinidius and trypanosome DNA was checked using PCR. The Chi-square test was carried out to determine whether there was an association between the presence of S. glossinidius and trypanosome infections. Out of the total tsetse flies collected, the prevalence of S. glossinidius and trypanosomes was 21.8% and 19.3%, respectively. The prevalence of S. glossinidius was 22.2% in Glossina morsitans and 19.6% in Glossina pallidipes. In relation to sampling sites, the prevalence of S. glossinidius was 26.0% in Chunga and 21.0% in Ngoma. DNA of trypanosomes was detected in 18.9% of G. morsitans and 21.4% of G. pallidipes. The prevalence of trypanosomes was 21.7% and 6.0% for Ngoma and Chunga, respectively. The prevalences of trypanosome species detected in this study were 6.4%, 4.6%, 4.0%, 3.7%, 3.1%, and 2.5% for T. vivax, T. simiae, T. congolense, T. godfreyi, T. simiae Tsavo, and T. b. brucei, respectively. Out of 63 trypanosome infected tsetse flies, 47.6% of the flies also carried S. glossinidius, and the remaining flies were devoid of S. glossinidius. A statistically significant association was found between S. glossinidius and trypanosomes (p < 0.001) infections in tsetse flies. Our findings indicated that presence of S. glossinidius increases the susceptibility of tsetse flies to trypanosome infections and S. glossinidius could be a potential candidate for symbiont-mediated vector control in these tsetse species.