A comprehensive data search was conducted across three electronic databases including (PubMed, Google Scholar, and CiNii Articles Incorporated Database) to identify articles and accompanying data reporting pathogens in bats, rodents, and NHPs. The search was restricted to studies published between January 1990 and December 2022. The period was selected in order to provide a comprehensive and nuanced understanding of the research trends and distribution of pathogens in the light of continuous changes in globalisation, pandemics, research tools national priorities, data availability, and demographic changes. Eligible original studies were identified using the following search terms with the help of Boolean operators (AND, OR): ((Virus) AND (Zambia)) AND (bat), ((Virus) AND (Zambia)) AND (rodent), ((Virus) AND (Zambia)) AND (non-human primates), ((Virus) AND (Zambia)) AND (baboons), ((Virus) AND (Zambia)) AND (Monkeys), ((Bacteria) AND (Zambia)) AND (bats), ((Bacteria) AND (Zambia)) AND (rodent), ((Bacteria) AND (Zambia)) AND (non-human primates), ((Bacteria) AND (Zambia)) AND (baboons), ((Bacteria) AND (Zambia)) AND (Monkeys), ((Protozoa) AND (Zambia)) AND (bats), ((Protozoa) AND (Zambia)) AND (rodent), ((Protozoa) AND (Zambia)) AND (non-human primates), ((Protozoa) AND (Zambia)) AND (baboons), ((Protozoa) AND (Zambia)) AND (Monkeys), ((Zoonoses) AND (Zambia)) AND (bats), ((Zoonoses) AND (Zambia)) AND (rodent), ((Zoonoses) AND (Zambia)) AND (non-human primates), ((Zoonoses) AND (Zambia)) AND (baboons), ((Zoonoses) AND (Zambia)) AND (Monkeys), ((Pathogen) AND (Zambia)) AND (bats), ((Pathogen) AND (Zambia)) AND (rodent), ((Pathogen) AND (Zambia)) AND (non-human primates), ((Pathogen) AND (Zambia)) AND (baboons), and ((Pathogen) AND (Zambia)) AND (Monkeys). The search strategy also included ((bats OR rodents OR “non-human primates” OR baboons OR monkeys) AND (bacteria OR protozoa OR zoonoses) AND (Zambia) AND (prevalence OR seroprevalence OR wildlife OR national park)) Following identification of some articles which specified certain pathogens, search terms were extended to include some names of identified pathogens as follows: ((bats OR rodents OR “non-human primates” OR baboons OR monkeys) AND (bacteria OR protozoa OR zoonoses OR Trypanosomes OR Giardia OR Cryptosporidium OR Coxiella OR Borrelia OR Paramyxovirus OR Leptospira OR Filovirus OR “Marburg virus” OR “Hepatitis virus” OR Rotavirus OR Arenavirus OR Rickettsia OR Babesia OR Anaplasma) AND (Zambia)).

Selected studies focused on rodents, bats, or NHPs in Zambia, reporting on sample size and type, diagnostic methods, and pathogen type. Studies had to be peer-reviewed, in English, fully accessible, conducted in Zambia, and published from January 1, 1990, to December 31, 2022. Experimental studies that combined both surveillance and experimentation were also included.

Studies were excluded if they lacked detailed methodological descriptions, did not report prevalence data, were not peer-reviewed, or were not written in English. Journal articles published outside the predetermined review period, and duplicates as well as non-original research were excluded from the review.

Data were independently extracted for each pathogen or potential pathogen by two reviewers (SMM and BM) using a standardised form for this review. It included animal species (rodent, bat, NHP), sample size, type, diagnostic methods, pathogen type, positives, and genetic matches via BLAST. Other data covered study location, design, publication year, authors, and journal. Discrepancies were resolved through discussion or a third reviewer who was available. Data were managed in Microsoft Office Excel 2018, ensuring integrity with backups. Zotero (Version 5.0.96.3) stored study details including titles, abstracts, authors, years, journals, and extracted PDFs where available, facilitating comprehensive information retrieval and management. The quality of the studies was evaluated using the JBI’s critical appraisal tools for prevalence and incidence studies to assess the trustworthiness, relevance, and results of the studies (30).

The prevalence rate of pathogens in selected animal species was directly extracted from the reviewed articles. For studies reporting data on multiple animal species, the prevalence was recalculated separately for each species of interest to ensure accurate representation (31–33). In instances where data from multiple studies were available, the cumulative prevalence of certain pathogens was determined by combining data from these studies. The public health risk associated with each reported pathogen was assessed based on existing evidence from the included articles and relevant literature demonstrating the pathogen’s potential to cause disease.

Descriptive data were summarised in tables showing the number of samples analysed, number of positive cases, and prevalence rates. Maps were created to show the distribution of sample collection sites across Zambia and reported highly infectious agents. A detailed summary of findings was presented, including a discussion and implications for future research.

All included studies were evaluated for adherence to ethical standards for animal research. This included reviewing whether the original studies obtained appropriate ethical approvals and consent for animal use.

The literature search identified 37 eligible articles. The number represents the actual number of surveillance studies conducted specifically on bats, rodents, and NHPs in Zambia from 1990 to 2022.

A total of thirty-seven original research articles were included in the analysis, covering the period January 1, 1990 to December 31, 2022. The search did not yield any articles within the investigated databases for the period 1990 to 2009. The highest publication frequency was observed in the years 2018 and 2019 (n = 10). When categorised by host type, 41%% (14/39) of the articles represented studies focused on bats (34–47), 30.8% (12/39) on rodents (23, 31, 48–56), and 25.6% (10/39) on NHPs (32, 57–65). One article (2.6%; 1/39) reported on both NHPs and rodents (33). In terms of pathogens detected, 66.7% (26/39) of the articles investigated viruses exclusively (31–34, 37–40, 43–47, 50–52, 55, 58–64), 17.9% (7/39) focused solely on bacteria (23, 35, 36, 41, 48, 53, 54), and 5.1% (2/39) protozoa alone (42). The remaining 10.3% (4/39) of the articles addressed mixed infections involving bacteria and viruses (49), helminths and viruses (56), as well as bacteria and protozoa (57).

Samples were collected from animals in study sites across 14 out of the 116 districts in Zambia (Figure 1). Reasons for selecting the reported study sites were not indicated. All the articles reported use of cross-sectional study designs. Short-term cross-sectional studies accounted for 94.9% (37/39) of all reported articles compared to 5.4% (2/37) of long term cross-sectional studies (38, 59). Almost all articles (97.4%, 38/39) had spleen, liver, kidneys, and blood as samples of choice with the exception of one study (2.6%, 1/39) in rodents that extended its investigations to semen (56).

Thirteen species of bats were reported within the study areas in Livingstone, Lusaka, Monze, Ndola and Serenje districts (34–47). The reported species of bats included Hipposideros gigas, Hipposideros vittatus, Miniopterus schreibersii, Rousettus aegyptiacus, Minipteros sp., Myotis sp., Rhinolophus simulator, Macronycteris vittatus, and other unknown species in the genera Rhinolophus and Hipposideros in Lusaka province where most of the investigations took place; Eidolon helvum in Ndola and Serenje (Kasanka National Park) districts; Epomophorus crypturus in Monze district; and Nycteris sp. in Livingstone district (34–47). In terms of common species, Rousettus aegyptiacus (23.2%, 719/3100) and Eidolon helvum (68.2%, 2114/3100) were the most captured species of bats, accounting for 91.4% (2,833/3100) of all reported bats.

Rodents belonging to 17 genera were identified in sampling sites in Chibombo, Kafue, Livingstone, Lusaka, Mazabuka, Mfuwe, Mpulungu, Namwala, Nyimba, Serenje, Sinda, and Solwezi districts. Reported rodent species included Acomys subspinosus, Aethomys chrysophilus, Arvicanthis sp., Cricetomys gambianus, Gerbilliscus leukogaster, Grammomys sp., Graphiurus sp., Hylomyscus alleni., Praomys sp., Lemniscomys rosalia, Mastomys natalensis, Mus minutoides, Otomys sp., Rattus rattus, Saccostomus campestris, Pelomys sp. (23, 31, 33, 48–53, 55). The distribution of rodents was fairly ubiquitous across the districts except for Mus minutoides which was only reported in Lusaka district. The most trapped rodent species was M. natalensis which accounted for 91.4% (1825/1996) of all captured rodents.

Chlorocebus and Papio were the only genera of NHPs reported in the reviewed articles (32, 57, 59–61, 64). The genus Chlorocebus was represented by Chlorocebus pygerythrus (vervet monkey) and Chlorocebus cynosures. Meanwhile, kinda yellow baboon (Papio kindae), yellow baboons (Papio cynocephalus), and Chacma baboons (Papio ursinus) comprised NHPs in the genus Papio. All species were reported in the sampling sites in Livingstone and Mfuwe districts except in the Kafue National Park where only Chlorocebus cynosures were sampled. The most captured NHP was P. cynocephalus (28.3%, 339/1198).

A combined total of 50 distinct infectious agents were reported in the reviewed articles. Bats and rodents each accounted for 36% (18/50) of all infectious agents whereas NHPs were responsible for 28% (14/50). Viruses were primarily the most frequently reported types of microorganisms accounting for 62% (31/50), mirroring the focus of the research in the reviewed articles. A total of 18% (9/50) of infectious agents were bacteria and the remaining 20% (10/50) were parasites. The array of infectious agents across bats, rodents, and NHPs represented highly infectious pathogens (Figure 2) and those with unknown zoonotic potential (Tables 1–4; Supplementary Table S1).

The 18 infectious agents reported in bats (Table 1) were primarily in Rousettus aegyptiacus and Eidolon helvum, comprising 77.8% (14/18) viruses, 16.7% (3/18) bacteria, and 5.5% (1/18) parasites. Noteworthy zoonotic viruses included EBOV, MARV, RVA, Borrelia faini, Leptospira sp., among others. In rodents, viruses and parasites each contributed 38.9% (7/18) to the reported infectious agents compared to 22.2% (4/18) by bacteria (Table 2). Most pathogens were reported in Mastomys natalensis. Notable pathogens were Yersinia pestis, Rickettsia felis, Coxiella burnetti, Calodium hepaticum, and Leptospira sp. Among NHPs, 71.4% (10/14) of the reported infectious agents were viruses (Table 3). Bacteria and parasites each accounted for 14.3% (2/14). Human parainfluenza virus type 3 (HPIV3), filoviruses, Trypanosoma brucei rhodesiens, and Anaplasma phagocytophilum were the headline pathogens.

All the articles included in the review utilized standard polymerase chain reaction (PCR) detection methods and reported the prevalence of pathogens, except for one study that did not (63). Sampling of non-human primates (NHPs) was predominantly focused on national parks in the Livingstone and Mfuwe districts, where the researchers had active projects. Additionally, the studies exhibited a bias toward viral pathogens, reflecting the specific interests of the researchers. Articles detailing zoonotic pathogen circulation in the animals of interest from 1990 to 2009 were absent. Although the reason for the absence of articles during that period could be linked to various factors, data from some review articles suggest a possible focus on anthrax, bovine tuberculosis, trypanosomiasis, tick-borne parasitic diseases, and other zoonoses in livestock (87–91). Therefore, there is need for further investigations over the observed trends in order to fully understand the evolution of research on zoonoses in Zambia. Nonetheless, post-2009, there was a notable surge in research involving bats, rodents, and NHPs (31–33, 35, 37, 39, 40, 49–52, 57, 60, 62). This increase in research activity likely stems from significant events such as the emergence of LUJV in Zambia in 2008 (1), the global swine influenza outbreak in 2009 (92), and the recurring, deadly outbreaks of Marburg virus disease (MDV) (70), and EVD in the neighbouring DRC (82).

Cross-sectional study designs were the most predominant, although they lacked subsequent follow-up research, except for two studies (37, 38). Therefore, the temporal dynamics related to abundance and likelihood of outbreaks of most reported pathogens remain poorly studied. These glaring gaps emphasise the critical need for robust long term surveillance studies in order to fully understand the spatial, temporal, and transmission dynamics of infectious agents within their micro-environments (56).

Understanding organ-specific pathogen distribution enhances targeted surveillance and diagnostic strategies, crucial for predicting and controlling infectious diseases. Most studies focused on spleen, liver, kidneys, and blood, with one exception using semen samples (56), highlighting their potential for pathogen detection, including viruses like EBOV and LASV (56, 93–95). This underscores the need to screen multiple organs and fluids to increase detection rates. For filovirus surveillance in bats, semen and seminal vesicles may offer valuable insights, as traditional samples (blood, liver, etc.) have scarcely yielded positive RNA results (14, 19). Integrating diverse sample types could enhance disease surveillance and response efforts significantly.

The distribution of pathogens mirrored the spread of their hosts. However, reservoir presence did not always correlate with infectious agents (51, 52, 56). Studies covered just 14 of 116 districts, notably neglecting Western and Luapula provinces with rich river systems ideal for diverse pathogens (56). This gap raises concerns about potential undetected zoonotic hotspots. Sparse surveillance hampered understanding of pathogen spread, crucial for guiding presumptive treatment in areas lacking multi-pathogen diagnostic capabilities in healthcare settings.

Serological and molecular evidence of bats and NHPs encountering pathogenic filoviruses raises significant public health concerns. Serological findings are notable as EBOV RNA has only been detected once in bats (19). EBOV and MARV cause deadly, unpredictable epidemics in Central and West Africa, with the 2014–2015 West African EBOV outbreak causing over 11,310 deaths (73, 96). In Zambia, spill-over risk stems from R. aegyptiacus bats, and migratory E. helvum bats from filovirus-endemic Congo Basin countries (97). Infected humans crossing the open border from the EBOV hotspot, DRC, are potential sources of human to human transmission (82). Wild RVAs (34, 44) also raise public health worries due to their potential impact on current vaccines (98), particularly the possible emergence of reassorted viruses not covered by existing vaccines (99). The discovery of an RVA with 98.1% nucleotide similarity to shows the potential cross species transmission between bats and humans probably through contaminated shared fruits and water (34). On the other hand, HPIV3, Hantavirus, Zika virus, and novel pathogens such as LPHV and LNKV cannot be ignored. Discovery of an HPIV3 sequence in baboons, closely resembling a strain isolated from a sick child in Saudi Arabia (60, 100), underscores the need for investigations into zoonotic HPIV3 in children in Zambia. Therefore, both known and novel viruses reported herein, demand rigorous investigation to mitigate their potential threat to local and global health.

Yersinia pestis, C. burnetii, A. phagocytophilum, and Leptospira sp. were the headline pathogens in the reviewed articles. Yersinia pestis poses a documented threat in Zambia, causing sporadic outbreaks of bubonic plague, particularly in eastern regions (23, 54). On the other hand, C. burnetii, responsible for Q fever which has never been reported in humans in Zambia, seems widespread within multiple rodent hosts in diverse ecological settings (101). Leptospira sp., similar to other zoonotic pathogens, suffers from a significant paucity of molecular and human data in Zambia. This calls for public health vigilance due to recent outbreaks in humans in neighbouring Tanzania (102). Anaplasma phagocytophilum was another significant zoonotic pathogen reported in wildlife. It is a multi-host intracellular pathogen, which causes varying severity of human febrile granulocytic anaplasmosis and encephalitis (103, 104). Like the emergence of B. faini which caused Zambia’s first clinical case of human borreliosis (36), its detection is alarming and warrants further investigations. The disease caused by B. faini presented with symptoms resembling malaria and flu, including high fever, initially confusing diagnosis at a local clinic, a common phenomenon with zoonotic pathogens (105). B. faini formed a monophyletic lineage akin to relapsing fever borreliae in the USA (36). Further investigations are critically needed to understand its overall impact on human health in Zambia.

Significant public health threats from pathogens including T. b. rhodesiense, C. hepaticum, T. spiralis, and T. ovis still exist as reported in the articles. Human African trypanosomiasis (HAT) caused by T. b. rhodesiense provided a problematic diagnostic process among clinicians, resulting in delayed treatment, suggesting need for refresher courses on this neglected zoonoses (65, 66). Strategic awareness campaigns may be helpful to educate the public about HAT and pathogenic parasites such as C. hepaticum, T. spiralis, and T. ovis (106–110).

In the context of Zambia’s open boarders and its role as a key transportation hub for Southern Africa, the implications of the existence of highly pathogenic zoonotic organisms with potential for major outbreaks are even alarming. It significantly heightens the risk of zoonotic pathogen outbreaks spreading rapidly across the region. The free international connectivity and high traffic can swiftly transform a local outbreak into a pandemic. Economically, the region could suffer from costly public health crises, disrupting trade and travel, and impacting regional stability similar to COVID-19 (1). To mitigate the risk of zoonotic disease spread through these transportation hubs, robust screening and surveillance systems are vital. Continuous and consistent aggregation and analysis of available data on circulating zoonotic pathogens can help map hotspots and enhance the country and region’s ability to respond effectively.