Current knowledge of vector-borne zoonotic pathogens in Zambia: A clarion call to scaling-up "One Health" research in the wake of emerging and re-emerging infectious diseases.

BACKGROUND: Although vector-borne zoonotic diseases are a major public health threat globally, they are usually neglected, especially among resource-constrained countries, including those in sub-Saharan Africa. This scoping review examined the current knowledge and identified research gaps of vector-borne zoonotic pathogens in Zambia. METHODS AND
FINDINGS: Major scientific databases (Web of Science, PubMed, Scopus, Google Scholar, CABI, Scientific Information Database (SID)) were searched for articles describing vector-borne (mosquitoes, ticks, fleas and tsetse flies) zoonotic pathogens in Zambia. Several mosquito-borne arboviruses have been reported including Yellow fever, Ntaya, Mayaro, Dengue, Zika, West Nile, Chikungunya, Sindbis, and Rift Valley fever viruses. Flea-borne zoonotic pathogens reported include Yersinia pestis and Rickettsia felis. Trypanosoma sp. was the only tsetse fly-borne pathogen identified. Further, tick-borne zoonotic pathogens reported included Crimean-Congo Haemorrhagic fever virus, Rickettsia sp., Anaplasma sp., Ehrlichia sp., Borrelia sp., and Coxiella burnetii.
CONCLUSIONS: This study revealed the presence of many vector-borne zoonotic pathogens circulating in vectors and animals in Zambia. Though reports of human clinical cases were limited, several serological studies provided considerable evidence of zoonotic transmission of vector-borne pathogens in humans. However, the disease burden in humans attributable to vector-borne zoonotic infections could not be ascertained from the available reports and this precludes the formulation of national policies that could help in the control and mitigation of the impact of these diseases in Zambia. Therefore, there is an urgent need to scale-up "One Health" research in emerging and re-emerging infectious diseases to enable the country to prepare for future epidemics, including pandemics.

Introduction

Zoonoses are infectious diseases that are spread from animals to humans or vice versa. It is estimated that more than 60% of emerging human infectious diseases have their origins in animal populations [1], with climatic shifts and anthropogenic land use changes implicated as major drivers of this emergence. These drivers lead to the reduction of the biogeographical distances between the vector and/or zoonotic pathogens or the infected animal populations and humans, allowing for pathogen transmission and subsequent human zoonotic infections [2,3]. In sub-Saharan Africa, zoonoses that have emerged in the last four decades are mostly viral and bacterial agents that have caused disease burdens of varying proportions in humans [2]. The impact of zoonoses on public health systems globally can be far reaching. More generally, it is estimated that more than 2 million human deaths and 2.4 billion cases of illness every year globally are attributed to zoonoses [4]; a situation requiring urgent intervention from the public-health and scientific communities.

Continued surveillance of zoonotic pathogens is crucial in building public health systems that translate into effective strategies for prevention and control of zoonotic diseases [5]. Ideally, this will include robust research infrastructure both in expertise and laboratory capacity as well as implementation of a “One Health” approach which should be multi-sectoral and multidisciplinary in nature as well as operationally [6]. Implementing such an approach will not only improve the response of public health systems in mitigating the impact of zoonoses, but will also benefit the animal, environmental and wildlife sectors which are critically important components of the economy [5]. The current challenges in the fight against zoonoses in resource-constrained countries including Zambia are to a large extent due to lack of appropriate diagnostics and limited knowledge about zoonotic pathogens, resulting in the misdiagnosis of conditions unfamiliar to medical practitioners. This is a reflection of insufficient knowledge of what pathogens are in circulation, compounded by an absence of appropriate diagnostics. It has been shown that across Africa, there tends to be under/misdiagnosis of less known infections accompanied by overdiagnosis of the most common ones [7].

In Zambia, several zoonotic pathogens have been reported in different ecological landscapes, but their etiological impact on human diseases remains unclear. Currently, information on the disease burden attributable to vector-borne and emerging or re-emerging zoonoses in humans is obscure. For example, despite a number of zoonotic pathogens (bacterial and arboviral zoonoses) being recognized as etiologic agents of febrile illnesses [8], they are not routinely screened for in the differential diagnosis of febrile illnesses in most healthcare facilities, largely due to lack of diagnostic capacity as well as a poor understanding of the local epidemiology of these pathogens among health practitioners.

Here, we reviewed studies reporting zoonoses or their etiological agents in Zambia focussing on mosquito-, tick-, flea- and tsetse-borne pathogens, and identified knowledge gaps that should be addressed in future studies. This study further aimed at stimulating interest in scaling-up research in emerging and re-emerging pathogens in order to create awareness among public health practitioners and drive policy change towards response, diagnosis and management of zoonotic diseases in the country. Currently, genomic surveillance of these emerging and re-emerging zoonotic pathogens in humans is hugely lacking as earlier studies were mostly serological. Thus, there is an urgent need to provide complementary genomic data necessary to inform and guide the response, diagnosis and management of zoonotic diseases in the country.

Methods

This review was based on publications indexed in the Web of Science, PubMed, Scopus, Google Scholar, CABI or Scientific Information Database (SID), prior to June, 2021. We also included a study “in press” conducted by the authors. Our online search included a combination of keywords such as–“mosquito-borne”, “tick-borne”, “flea-borne”, “tsetse-borne” and/or “zoonoses in Zambia”, “Vector-borne disease” and “arboviral diseases in Zambia” and filtering the literature to include only publications describing their occurrence in Zambia. We further searched these electronic databases for literature reporting the occurrence of these diseases in animals and humans in Zambia, Southern Africa and in some instances at a continental level to build context for the regional perspective. Finally, we expanded our search to review references cited in the searched publications to increase coverage of the literature that was relevant to the preparation of this review. Through this filtering process, a total of 39 articles reporting detection of vector-borne zoonoses in Zambia were identified for the preparation of the review. Four main categories for this review were prepared to highlight mosquito-borne, tick-borne, flea-borne and tsetse-borne zoonotic pathogens reported in Zambia and further discussed to identify research gaps to inform future investigations.

Results

Mosquito-borne Zoonotic viruses

Mosquito-borne viruses of medical and veterinary importance that have been reported in Zambia mainly belong to four virus genera; Flavivirus, Alphavirus, Orthobunyavirus and Phlebovirus.

Flaviviruses

The genus Flavivirus consists of positive single-stranded RNA enveloped viruses. Notable members of public health significance include; Yellow fever virus (YFV), Ntaya virus (NTAV), Dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), Japanese encephalitis and several other viruses which mostly cause febrile and neurological manifestations in humans [9].

Yellow fever virus (YFV), the prototype virus in the genus Flavivirus, is a re-emerging threat with more than 508 million people at risk of infection in tropical African countries where the virus remains endemic [10]. The virus is mainly transmitted by Haemagogus and Aedes mosquitoes either through the sylvatic or urban cycles of transmission [11]. Clinically, YFV infection is characterized by fever, headache, jaundice, muscle pain, nausea, vomiting and fatigue [11]. The earliest mention of YFV in Zambia dates back to the early 1940s from seroprevalence studies that revealed low exposure of people in present-day North-Western province [12]. More recently, a study in Western and North-Western provinces conducted in 2015 revealed a seroprevalence of 0.2% (3,325 study participants), suggesting a considerably low active circulation of the virus [13].

Ntaya virus (NTAV) was first isolated in western Uganda in the early 1950s [14]. Though a limited number of NTAV infections have been recorded across the globe, notable consistent clinical symptoms observed in humans include fever, headache and neurological symptoms [15]. Though not confirmed, Culex mosquitoes are suspected to be vectors of NTAV [16]. Serological evidence of NTAV in Zambia was documented in 1977 among international travellers that visited Zambia [15]. This study is the only report of the pathogen in the country. Elsewhere, serological evidence of infection has been reported in migratory birds as well as farmed animals, suggesting a potential reservoir role of these hosts [17,18].

Dengue virus has in the recent past been viewed as one of the most serious re-emerging vector-borne zoonosis with more than 390 million infections per year in the Americas, tropical Africa and Asia [19]. The virus is transmitted by mosquitoes of the genus Aedes [20]. Infections in humans can either be asymptomatic or symptomatic. In apparent and mild clinical infections, they are characterised by headache, rash, myalgia, arthralgia, and fever. Hepatitis, neurological disorders, myocarditis, and shock have been observed in severe infections [21]. Serological evidence of DENV in Zambia was first reported among European expatriates and travellers that had visited Zambia between 1987 and 1993 [22]. Further reports of DENV were in North-Western and Western provinces of Zambia with a prevalence rate of 4.1% (149/3624) [23], and more recently in Central Province [24]. However, there are currently no reports of human clinical cases.

Sylvatic and urban cycling exist for ZIKV transmission, although in Africa, sylvatic cycle seems to be the main transmission route with mosquitoes of the genus Aedes being the principal vectors [25]. Clinically, human infections are characterised by mild symptoms of fever, rash, joint and muscle pain [26]. In pregnant women, the infection can lead to microcephaly in the infants [26]. To date, there are no documented human clinical cases of ZIKV infection in Zambia. However, its possible presence in Zambia has been demonstrated through serosurveys with reported prevalence rate of 6% (217/3625) in humans in Western and North-Western provinces [27], and 10.8% (23/214) in Lukanga swamps of Central Province [24]. In addition, neutralizing antibodies against ZIKV were detected in 34.4% (33/96) of African green monkeys and baboons inhabiting different ecosystems in Zambia, a finding that was indicative of a recent active infection and possible sylvatic maintenance of the pathogen in the country [28].

Generally, WNV infections in humans are predominantly asymptomatic. However, fever, neurological disease and death have been reported in severe symptomatic cases [29]. The first report of WNV in Zambia was in 2015 when a seroprevalence study conducted showed a prevalence rate of 10.3% (370/3625) for WNV among humans in North-Western and Western provinces [30]. Genetic evidence of the circulation of the virus has also been shown in Culex quinquefasciatus mosquitoes collected in Western Province [31] as well as in crocodiles [32] with lineages 2 and 1a reported in the respective studies. The lineage 2 strain reported in Zambia was shown to be closely related to strains circulating in South Africa, which have been reported to be associated with neurological disease in humans [31].

Alphaviruses

Alphaviruses are enveloped, positive-sense RNA viruses with all human infective viruses being mosquito-borne. A notable member in this group is chikungunya virus (CHIKV) that causes chikungunya fever. It is an emerging mosquito-borne disease that is principally transmitted by Aedes mosquitoes [33]. The disease shares many clinical similarities with DENV including acute onset of fever, joint pains, headache and fatigue. Previously, chikungunya fever was considered a self-limiting disease. However, severe complications, including acute viral hepatitis and death, have been reported in elderly patients and people with chronic comorbidities [34]. The first suspected reports of chikungunya fever in Zambia were recorded in the colonial era on the Copperbelt Province along the Kafue River basin with most cases having classical symptoms of CHIKV infection coupled with generalised superficial lymphadenopathy [35]. However, in 1999, antibodies against Sindbis virus (SINV) were detected in 0.1% (1/670) of German travellers that had spent at least 4 months in Zambia [36], indicating the possible presence of this pathogen in the country. Recently, a study conducted in 2016 among residents of Lukanga swamps in Central Province, revealed a 36.9% (79/214) sero-positivity for CHIKV and 19.6% (42/214) for Mayaro virus (MAYV) [24]. Interestingly and in contrast, a recent study that screened for alphaviruses using 9,699 mosquitoes collected from other geographical areas in Zambia (2014–2017) could not detect known alphaviruses (CHIKV, O’nyong-nyong virus (ONNV), SINV etc.) [37]. One possible explanation for this discrepancy were sampling sites for the vector-based study that did not include Lukanga swamps. It is also possible that the species composition of the tested mosquitoes may have contributed to the discrepancy. For instance, the majority of the captured mosquitoes were Culex and alphaviruses such as CHIKV and O’nyong-nyong virus (ONNV) could be missed as their principle vectors are Aedes and Anopheles mosquitoes, respectively. Nevertheless, it is imperative that future genomic surveillance studies should also target to screen alphavirus vectors in Lukanga swamps where human exposure to these pathogens has been documented.

Phleboviruses

Rift Valley fever virus (RVFV) infections in humans are characterised by a wide range of symptoms, which may include fever, myalgia, arthralgia, headache encephalitis, haemorrhage, hepatitis, and ocular pathology/retinitis [38]. Human zoonotic infections are usually preceded by the disease in wild and domestic animals [38]. In Zambia RVFV was first reported in humans in Chisamba District, Central Province, in 1984 where it caused some deaths [39]. Further evidence of the disease was provided in subsequent studies conducted in the early 1990s that indicated that cattle-dominated regions with higher amounts of rainfall had high seropositivity in the herds tested [39,40]. However, for the past three decades, no reports of outbreaks have been noted in humans or animals defying the epidemic resurgence pattern of 10–15 years as previously proposed [41]. However, a recent study suggests silent circulation of RVFV in cattle herds [42] corroborating with evidence suggesting that the lack of natural physical barriers between Zambia and other countries in the region that have reported outbreaks in the recent past, cross-border livestock trade and the presence of competent vectors present a real threat of RVFV in Zambia [43-45].

Tick-borne zoonotic pathogens

Viruses

Notable tick-borne viral zoonoses include tick-borne encephalitis virus (TBEV) and Crimean–Congo haemorrhagic fever virus (CCHFV), with CCHFV being the most recognised tick-borne viral zoonosis globally. Infections due to CCHFV in humans are characterised by headache, fever, joint pain, stomach pain, and vomiting [46]. Currently, there is only a single report of CCHFV in Zambia, with IgG antibodies being detected in 8.4% (88/1,047) of cattle [47]. The pathogen genome was detected in 3.8% (11/290) of Hyalomma ticks, the principal vector of CCHFV [47]. Genomic analyses revealed that one of the detected viruses was a genetic reassortant between African and Asian strains, intimating possible dissemination of CCHFV across continents. There is currently no report of tick-borne viral infections in humans in Zambia.

Bacteria

A number of tick-borne bacterial fever-inducing pathogens of zoonotic significance have been described to circulate in different hosts and vectors in Zambia. These belong to bacterial families of Rickettsiaceae, Anaplasmataceae, Spirochaetaceae and Coxiellaceae. Clinically, symptoms associated with tick-borne bacterial zoonoses are usually non-specific, making their diagnosis and treatment challenging and can include; fever, headache, body rash, nausea, joint and muscle pains [48].

In a study targeted to screen zoonotic pathogens in free-ranging nonhuman primates (NHPs) inhabiting various ecological landscapes in Zambia, zoonotic Anaplasma phagocytophilum was molecularly detected in 13.6% (12/88) of samples screened [49]. In another study, Vlahakis et al., (2018) [50], reported the first molecular identification and characterization of canine-associated zoonotic Anaplasma species in Zambia. The prevalence rate was 9% (27/301) for the dog population screened [50]. In addition, Ehrlichia canis and Anaplasma platys were genetically detected in dogs screened in Lusaka province [51].

Rickettsia species that have been identified in ticks and NHPs thus far in Zambia include R. africae, R. raoultii, R. conorii, R. massiliae, R. lusitaniae, R. hoogstraalii, R. aeschlimannii-like, and some unidentified Rickettsia species [52-56]. Notably, most of the Rickettsia species detected in the country are known human pathogens. Though no human clinical cases have been reported so far, serological evidence for infection with R. conorii and R. typhi was reported in 1999 with prevalence rates of 16.7% (63/377) and 5.0% (19/377), respectively [57].

Human borreliosis is characterised by erythema migrans, fever, chills, headache, fatigue, muscle and joint aches, and swollen lymph nodes. Only one case of human relapsing fever caused by Borrelia spp. has been documented in Zambia involving a patient with a history of tick bite from a soft tick (Ornithodoros faini) known to infest cave dwelling fruit bats (Rousettus aegyptiacus) [58]. The isolated Borrelia spp. was more closely related to New World relapsing fever Borreliae, indicating the possibility of these zoonotic infections being imported from afar places, possibly through migratory bats. Despite being a single case of human borreliosis, these results suggest that regions in Zambia with overlapping ecological landscapes of O. faini ticks and R. aegyptiacus bats may be at risk of transmission of tick-borne relapsing fever to humans [58].

Coxiella burnetii, the causative agent of Q fever in humans is a zoonotic agent with ubiquitous global distribution. Symptoms in human infections include; high fever, chills or sweats, a cough, chest pain while breathing, headache, diarrhoea, nausea, abdominal discomfort and in some instances jaundice. It has been detected in a number of tick species known to infest livestock such as Rhipicephalus, Amblyomma and Haemaphysalis [59]. It was first recognised in Zambia among livestock (cattle and goats) in Chama, Chongwe and Petauke districts through molecular typing [60]. More recently, Chitanga et al., (2018) provided the first genetic evidence of C. burnetii circulating in Zambian dogs and rodents [61]. In humans, about two decades ago, serological evidence of Q fever infection was demonstrated in Zambia with a prevalence rate of 8.2% [57,62]. These studies show that the risk of transmission of C. burnetii to humans especially among livestock farming communities and pet owners is conspicuous and presents a real but unappreciated threat [63].

Flea-borne zoonotic pathogens

Flea-borne zoonoses are emerging or re-emerging worldwide, and their incidence is on the rise. Furthermore, their distribution and that of their vectors is shifting and expanding [64]. However, their public health significance in Zambia has not been sufficiently elucidated partly due to lack of epidemiological surveillance despite the abundance of cosmopolitan flea vectors. To date only Yersinia pestis and Rickettsia felis have been reported in Zambia [65-67].

Recognized for its previous global pandemics called “Black death”, Y. pestis was first documented to cause human infections in Zambia in 1914 with a number of subsequent outbreaks recorded [68]. Bubonic plague is the most common disease form of the plague in Zambia, and is a rodent-flea-borne re-emerging zoonosis characterised by acute onset of fever, headache and body malaise. In Zambia, it is endemic in Namwala (Southern Province), Sinda and Nyimba districts (Eastern Province). A study conducted between 2015 and 2016 in Sinda and Nyimba districts showed the circulation of Y. pestis among multiple hosts including humans, fleas, shrews and rodents [69]. Sequences of strains isolated from these hosts were similar, indicating a multi-host zoonotic circulation and they demonstrated very close evolutionary relationships with Antiqua strains from the Democratic Republic of Congo and Kenya. In addition, serological studies conducted in the same area showed that 16.0% (4/25) of rodents, 16.7% (1/6) of shrews and 6.0% (5/83) of goats were positive for IgG antibodies against Fraction 1 antigen of Y. pestis, a result supporting the idea of active circulation of the bacterium in the geographic area [70]. Risk factors associated with human outbreaks in Zambia included heavy rains leading to a huge surge of rodents and fleas, hunting, unhygienic human habitats, preparation and consumption of rodents [71,72].

Flea-borne spotted fever caused by Rickettsia felis is one of the neglected emerging rickettsioses. Cat fleas have been considered the hosts and biological vectors of R. felis with dogs and cats acting as mammalian reservoirs [73,74]. R. felis is transovarially and transtadially maintained in cat fleas [75]. R. felis is classified in the spotted fever group (SFG) rickettsiosis based on clinical disease manifestation. The first human case of R. felis infection was reported in 1994 in Texas, USA [76]. Since then, it remained neglected until it emerged as a cause of febrile illness in sub-Saharan Africa [77]. In Zambia, the bacterium was recently detected in 4.7% (7/150) of dogs and 11.3% (12/106) of Mastomys species of rodents [67]. Additionally, 3.7% (2/53) of the sampled cat fleas collected from dogs were positive for R. felis [67]. Another potential cause of flea-borne rickettsiosis in humans is R. asembonensis, which is similarly transmitted from fleas to humans. Although its pathogenicity in humans is unknown, several reports indicated the infection is associated with acute febrile illness [78]. The prevalence of R. asembonensis in cat fleas collected from Zambian dogs was reported to be high showing 41.5% (22/53) [67]. Importantly, the DNA of R. asembonensis was also detected in two human blood samples from Zambia out of 1,153 screened. This suggests possible human infection by R. asembonensis possibly through cat flea bite [79].

Tsetse-borne pathogens

Trypanosomiasis has been endemic in Zambia for more than a century and is prevalent in wildlife-livestock ecosystems having geographical overlaps with tsetse-belts, where the fly that vectors these pathogens are found [80]. The causative agent, Trypanosoma sp., is the only pathogen transmitted by the biting tsetse flies (Glossina spp.) that are endemic to the Luangwa and Zambezi valleys and Kafue National Park (KNP) [80]. Of public health concern in Zambia is the human infective trypanosome T. b. rhodesiense which is endemic in Eastern and Southern Africa in various animal hosts and tsetse flies [81]. Recently, Squarre et al., (2020), detected several Trypanosoma sp. in multiple wildlife species inhabiting KNP including the human infective T. b. rhodesiense [82]. The zoonotic trypanosome has also been reported in both indigenous and exotic dog breeds kept as pets in areas bordering endemic zones, a finding showing the increased possibility of transmission of Human African Trypanosomiasis (HAT) from the wildlife-livestock-tsetse interface to surrounding human communities [83-85]. Evidence is increasing to suggest that more cases of HAT than previously thought occur among communities lying along the tsetse belts. For example, in 2012, HAT was diagnosed in four patients from the Luangwa and Zambezi valleys that presented with febrile illness [86]. Furthermore, a severe case of HAT was also recorded in 2016 in KNP from a patient that presented with persistent fever [87]. Whilst these documented cases may seem to be sporadic, previous passive surveys for HAT indicated that HAT was frequently reported among communities or workers living in tsetse fly-infested wildlife protected areas but was underreported [88,89]. Among the reasons for this underreporting included lack of diagnostic capacity in rural health centres, low awareness levels among health care providers, and frequent misdiagnosis with familiar diseases such as malaria [90]. This further affirms that HAT is still a neglected tropical disease in Zambia [91].

Pathogen discovery

On the pathogen discovery front, some strides have been made in that a number of novel arboviruses have been detected in the country. Among them include the tick-borne phlebovirus, Shibuyunji virus, which was detected from Rhipicephalus ticks in Shibuyunji and Namwala district [92,93]. Additionally, Harima and colleagues (2021), characterised the Mpulungu flavivirus, a novel tick-borne flavivirus isolated from a Rhipicephalus muhsamae tick with a typical vertebrate genome signature, suggesting its potential to infect vertebrate hosts [94]. However, studies in both humans and animals associated with the ticks are certainly needed to clarify the veterinary and public health importance of these novel tick-borne viruses. Others include Mwinulunga alphavirus isolated from Culex quinquefasciatus mosquitoes [37] and the newly identified Barkedji-like virus; a novel flavivirus detected from Culex spp. mosquitoes [95]. Current experimental data so far suggests that both Mwinilunga alphavirus and Barkedji-like virus fail to replicate on vertebrate cell lines, but it would be interesting to know the implications of this experimental data on host range restriction and whether they have any zoonotic potential [37,95].

Identified research gaps on vector-borne zoonotic pathogens in Zambia

For mosquito-borne pathogens, we observed that most reports (14/19; 73.6%) were in humans, with only five (26.4%) other studies reporting the pathogens in vectors or other mammalian hosts (Table 1). In addition, the majority of the studies were serological in nature (17/19; 89.5%), except for two (10.5%) studies that reported genetic detection of WNV from mosquitoes and crocodiles (Table 1). This points to the need for increased genomic surveillance of mosquito-borne pathogens in vectors, animals and humans for a better understanding of the molecular epidemiology of these diseases (Table 2). Considering the limited number of reports documenting clinical cases, establishment of facility-based surveillance of vector-borne zoonoses in health facilities could provide valuable insights on disease severity, mortalities associated with infections and immune responses. As such, the need for building laboratory capacity for routine screening of these pathogens as well as training laboratory personnel cannot be overemphasised. Vector ecology studies aimed at understanding the drivers of vector abundance, pathogen host range (i.e., including the range of vectors and reservoirs), parasite-host interactions and factors influencing frequency of human-vector contacts should be prioritized. Remote sensing and risk-based mapping are other research areas that are needed to inform appropriate interventions of vector-borne zoonoses.

Table 1

Studies reporting the serologic and/or molecular detection of vector-borne pathogens either in vectors and/or animals including humans in Zambia.

Vector-borne Pathogen	Study carried out in:
	Vectors	Animals	Humans
Mosquito-borne
Yellow fever virus (YVF)			#12,13
Ntaya virus (NTAV)			#15
Dengue virus (DENV)			#23,24
Zika virus ZIKV)		#28	#24,27
West Nile virus (WNV)	†31	†32	#30
Chikungunya virus (CHIKV)			#24,35
Mayaro virus (MAYV)			#24
Sindbis virus (SINV)			#36
Rift valley fever virus (RVFV)		#39,40,42
Tick-borne
Crimean–Congo haemorrhagic fever virus (CCHFV)	†47	#47
Anaplasma spp.		†49,50,51
Ehrlichia spp.		†51
Rickettsia spp	†52,53,54,55	†56	#57
Borrelia spp	†58	†58	†58
Coxiella burnetii		†60,61	#57
Flea-borne
Yersinia pestis	†65,69,70	‡70	†69
Rickettsia felis	†67	†67
Rickettsia asembonensis	†67		†79
Tsetse-borne
Zoonotic Trypanosoma sp		†82,83,84,85	†86,87

Notes: The † symbol in the table denote molecular detection; the # symbol denote serologic detection and the ‡ symbol denotes studies with both molecular and serologic detection. The numbers next to the symbols are reference list numbers of studies that reported the detection of vector-borne pathogens in Zambia.

Table 2

Research priority areas for vector-borne zoonoses in Zambia requiring research funding.

Vector-borne zoonoses	Research priority areas
	Surveillance	Diagnostics	Capacity-building
Mosquito-borne	• Increased genomic surveillance of pathogens in vectors, reservoir hosts, companion animals and domestic livestock to improve the molecular epidemiology comprehension and development of control strategies.• Increased serological surveillance covering new geographic areas where competent vectors exist.• Increased clinical surveillance of the pathogens in known endemic regions.• Vector ecological studies to inform their distribution	• Utilization of metagenomics next generation sequencing for detection, identification and characterization of known and novel pathogens in clinical specimens, mosquitoes, wild and domestic animals.• Development and deployment of affordable viral antigen capture rapid diagnostic kits for use in clinical settings• Improvement of sensitivity and specificity of serological assays	• Capacity building for improved and routine diagnostic screening in public healthcare facilities.• Capacity building for remote sensing, risk-based mapping and modelling to support vector ecological studies• Capacity building in genomic surveillance and bioinformatics for pathogen detection, identification and characterization
Tick-borne	• Serological, genomic and clinical surveillance of viral tick-borne pathogens in humans and other reservoir hosts.• Serological, genomic and clinical surveillance of bacterial tick-borne pathogens in humans and other reservoir hosts.• Genomic surveillance of pathogens in competent vectors• Molecular epidemiological studies to inform control strategies• Vector ecological studies to inform their distribution	• Utilization of metagenomics next generation sequencing for detection, identification and characterization of known and novel pathogens in clinical specimens, vectors, wild and domestic animals.• Development of low-cost multiplex molecular tools for detection of tick-borne pathogens of veterinary and medical importance.	• Capacity building for improved and routine diagnostic screening in public healthcare facilities.• Capacity building for remote sensing, risk-based mapping and modelling to support vector ecological studies• Capacity building in genomic surveillance and bioinformatics for pathogen detection, identification and characterization
Flea-borne	• Heightened awareness about risk factors for flea-borne zoonoses among high-risk populations.• Vector ecological studies to inform their distribution	• Development and deployment of affordable real-time PCR assays for detection of multiple genes of Rickettsia in clinical and vector specimens, thus negating the need for sequencing for pathogen identification• Utilization of metagenomics next generation sequencing for detection, identification and characterization of known and novel pathogens in clinical specimens, vectors, wild and domestic animals.	• Capacity building for improved and routine diagnostic screening in public healthcare facilities.• Capacity building for remote sensing, risk-based mapping and modelling to support vector ecological studies• Capacity building in genomic surveillance and bioinformatics for pathogen detection, identification and characterization• Establish specific reference laboratories for detection and identification of flea-borne pathogens.
Tsetse-borne	• Serological and genomic surveillance in humans in endemic areas• Continued surveillance in wildlife, livestock, and pets in tsetse-infested regions• Vector ecological studies to inform their distribution• Establishment of a national Human African Trypanosomiasis surveillance and tsetse control programs	• Validation and utilization of molecular techniques (e.g. PCR and LAMP) which are more sensitive than microscopy for passive case detection in primary care centres.• Application of molecular techniques, including next generation sequencing for pathogen detection, identification, and characterization from vectors, livestock, pets and humans.	• Development of laboratory and healthcare personnel capacity for improved and routine diagnostic screening in public healthcare facilities, particularly in tsetse-infested regions.• Capacity building for remote sensing, risk-based mapping and modelling to support vector ecological studies• Capacity building in genomic surveillance and bioinformatics for pathogen detection, identification and characterization

Discussion

This review sought to examine the current knowledge of vector-borne zoonotic pathogens in Zambia. Overall, the study found considerable evidence of presence and active circulation of these pathogens among vectors, wildlife and domestic animals and humans. Strikingly, reports of human clinical cases attributable to vector-borne pathogens were very limited. This finding could suggest a lack of diagnostic capacity for detection and identification of these infections in many health facilities in the country, a situation that has derailed a better appreciation of the epidemiological picture of vector-borne zoonoses in Zambia, as well as in many African countries [5].

The studies on mosquito-borne viruses revealed that North-Western, Western, Central, Copperbelt and Southern provinces of Zambia could be hotspots for human infections and should be targeted for both field and facility-based surveillance. In some areas, several mosquito-borne viruses were detected [24,27]; this co-circulation has the potential to lead to co-infections, thus presenting a challenge for diagnostic and treatment options in the event of clinical disease as observed elsewhere [96]. With several outbreaks of mosquito-borne viruses being reported in Africa and Southern Africa in particular, along with overlapping ecological niches for the vectors, coupled with the serological and genetic evidence that has accumulated so far, the threat of mosquito-borne arboviral epidemics in Zambia seems possible [10,19,26,35,44,97].

Many studies reviewed in the present study were serological and there was evidence of co-circulation. However, considering that human arboviral infections especially flavivirus infections induce the production of cross-reactive antibodies, often making serology inconclusive, it is important that results from these studies are interpreted cautiously. In fact, this is further complicated by a patient’s previous flavivirus exposure, particularly in regions where multiple antigenically related flaviviruses co-circulate and makes it difficult to attribute human disease burden to specific flaviviruses [98,99]. We therefore strongly recommend for increased genomic surveillance of these pathogens to provide precise molecular epidemiological data that will complement the serological data. These data will be necessary to drive enactment of relevant policies that will tackle vector-borne diseases in Zambia. For instance, to be cost effective, the genomic surveillance could utilize banked sera in hospitals or residual blood specimens from routine diagnostic screening of other diseases and could use consensus screening tools such as multiplex PCRs to increase on detection rates. The genomic surveillance could also target companion animals and livestock sharing habitats with humans and essentially using them as sentinels for arboviral circulation in human populations.

With regards to risk factors, exposure of humans to vectors was the main risk factor associated with mosquito-borne arboviral infections reported in Zambia [13]. This included exposure to suitable breeding microhabitats for mosquitoes that are recognized as arboviral vectors. For instance, Aedes spp. mosquitoes are known to inhabit flowering pots around homes, water holding containers or unattended containers [100]. This phenomenon in Zambia was shown by Masaninga et al., (2016) who collected vector mosquitoes (Culex and Aedes spp.) from outdoor containers, in urban regions of North-Western and Western provinces of Zambia where some zoonotic arboviruses have been documented through serological studies [101]. Humans are usually unaware of such microhabitats and this may enhance or sustain the arboviral infections as the vectors are sustained by suitable breeding habitats as well as sufficient blood-meals from humans. In this regard, awareness efforts are fundamental and could focus on educating communities on the impact of such microhabitats. In addition, economic activities such as fishing were strongly associated with high serological positivity for viruses such as CHIKV in humans [24]. Furthermore, high rainfall was linked to RVFV distribution in Zambia as previously observed in Mozambique and South Africa [40,44,45].

Recently, compelling evidence of circulation of CCHFV in cattle and ticks in Zambia was provided [47], supporting the idea that CCHFV is endemic in most Southern African countries and is the most recognised tick-borne viral zoonoses globally [46]. In fact, most neighbouring countries of Zambia are endemic to CCHFV [46]. With the presence of hard ticks in Zambia and the overlapping ecological habitats of these ticks across countries, the movement of people across the neighbouring countries may further exacerbate the zoonotic risk and establishment of CCHFV in the local ticks. [102]. It is important for future studies to now focus on investigating whether CCHFV circulates in humans, and even more so in areas where the vector ticks are endemic (Table 2).

We noted several reports of tick-borne bacterial zoonoses in livestock, pet dogs and wildlife. Though no human cases have been recorded, the detection of zoonotic Rickettsia and Anaplasma in NHPs and domestic dogs is concerning. Sequences of the detected Rickettsia in NHPs were similar to the Rickettsia sp. causing African tick bite fever in sub-Saharan Africa [49,103]. In addition, Rickettsia sp. were recently detected in ticks collected from cattle in southern Zambia suggesting the wide spread presence of the bacterium for African tick bite fever in Zambia [55]. Furthermore, the detected Anaplasma sp. (A. phagocytophilum and A. platys) from NHPs and dogs are known causative agents of human anaplasmosis [104]. Taken together, these reports suggest the active circulation of agents of African tick bite fever and human anaplasmosis in the sylvatic and urban cycles with a high potential to infect humans. Human rickettsiosis and anaplasmosis are emerging tick-borne zoonoses globally, causing fever related illnesses that are usually difficult to diagnose as most healthcare providers are unaware of them coupled with limited diagnostic capacity in most healthcare facilities of African countries [48]. Equally, the surveillance of C. burnetii should be scaled up in humans. From the reports we reviewed, we observed that the pathogen was detected in livestock, rodents and dogs, increasing the risk for human infection due to relative closeness of these animal hosts to humans. However, it remains unknown whether there is an active zoonotic transmission going on between the infected livestock and humans, as genome detection and clinical disease in humans has never been reported. Though only serological evidence of human infection with C. burnetii has been reported in the country, the infection is common with high prevalence rates and has been detected in a wide range of animal species across Africa [105].

Among the flea-borne pathogens reported in Zambia, the epidemiology and ecology of Y. pestis in Zambia appears to be fairly well known, though strategies on outbreak prevention and control still remain unclear. On the other hand, R. felis epidemiology requires elucidation of possible human infections as well as identification of endemic regions in order to devise effective control measures. Additionally, R. asembonensis, a close relative to R. felis has been reported in cat fleas collected from dogs as well as in human blood [79]. Further epidemiological studies are still necessary for the understanding of flea-borne zoonoses in Zambia. This is in a bid to put in place effective control measures as well as inform public health policies to curtail the emergence of flea-borne epidemics.

From the few HAT cases that has been reported so far, it is evidently clear that HAT is a neglected zoonosis in Zambia. Most of the earlier studies focussed more on the distribution of the vector and associated ecological factors. On the disease part, many studies done in Zambia and elsewhere [81] focussed more on African Animal Trypanosomiasis due to its associated economic losses neglecting the zoonotic potential of trypanosomes adapted to cause human disease. Only three reports were found to be describing HAT in humans based on passive surveillance [86,87,89], pointing to the lack of deliberate policies to improve diagnosis of HAT in the Zambian healthcare system. Concerted efforts are therefore required from all key players to ensure that active surveillance of human infective trypanosomes is mounted in order to improve the detection of HAT in Zambia. In fact, the detections of zoonotic trypanosomes in pet dogs compounds the problem of this neglected tropical disease as indicated by the ease of transmission from the sylvatic cycle to the urban cycle and requires timely interventions [83-85].

We also noted with concern the lack of data on vector control policies aimed at combating vector-borne zoonoses in Zambia. The mosquito control programme that has been ongoing for the past two decades is mainly indoor residual spraying and long-lasting insecticidal nets (LLIN) approaches targeting mosquito species transmitting malaria [106]. Though beneficial indirectly, to a large extent, it excludes other outdoor mosquito vectors that transmit vector-borne zoonoses. Similarly, tick and tsetse control in Zambia has been implemented in light of controlling livestock diseases [107,108]. In addition, flea control is largely non-existent in Zambia except perhaps through spraying of pets for ectoparasite control at individual household level. Indeed, the design of such vector control strategies has been one without a One Health framework in mind leaving vector-borne zoonoses unattended to from the vector control perspective. From the above, it is evident that there is an urgent need to develop national vector control policies and frameworks that will specifically target the control of vector-borne zoonoses in Zambia.

In addition, investment into studies such as remote-sensing based risk mapping has also the potential to improve the understanding of all the above-mentioned vector-borne zoonoses in Zambia. Such technology will help estimate how environmental variables such as humidity, precipitation, temperature and ground cover influence vector abundance, infection rates and interspecies transmission [109]. For example, changes in climate have been associated with increased transmission of vector-borne diseases [2]. Furthermore, it is widely recognized that vectors are sensitive to changes in temperature and precipitation [110]. Similarly, as discussed by Mills et al., (2010), variations in climate play a role in the emergence of vector-borne zoonoses because as the population density of vectors increases, the frequency of contact between vectors, humans or other hosts will also increase [111]. Therefore, application of remote-sensing based risk mapping will be critical in understanding the influence of climate change in the emergence of vector-borne zoonoses in Zambia (Table 2).

Conclusions

Overall, this review has shown that diagnosis of vector-borne zoonotic infections in Zambian healthcare facilities is very limited, if any. Most reports in humans were serological in nature and potentially limited in their interpretation to infer actual disease burden in humans owing to the cross-reactivity of arboviruses. In contrast, molecular as well as serological studies have shown the circulation of these pathogens in vectors and/or wildlife and domestic animals and suggest that infections and possibly clinical disease in humans could be going on undetected due to limited laboratory diagnostic capacity. Speculatively, it is our considered opinion that there is low awareness of emerging and re-emerging zoonoses in Zambia among healthcare practitioners. With glaring evidence provided in this review on the active circulation and transmission of vector-borne zoonotic pathogens across various invertebrate and vertebrate hosts, the need for establishment of policy frameworks towards tackling neglected vector-borne zoonoses cannot be overemphasized. It is our considered opinion that deliberate policies for laboratory strengthening to support routine screening of these pathogens in health facilities are urgently needed for improved diagnosis and management of zoonoses in the country.

De facto, scaling up of research into emerging and re-emerging zoonotic pathogens will form the basis for informing these interventions and addressing the existing knowledge gaps. Hence, there is an urgent need for the research community in Zambia and its cooperating partners to urgently scale up One Health research on vector-borne and emerging and re-emerging zoonoses not only to inform policies that could translate into improved healthcare provision, but also enable the country to prepare for future epidemics, including pandemics.

12 Oct 2021

Dear Dr Simulundu,

Thank you very much for submitting your manuscript "Current knowledge of vector-borne zoonotic pathogens in Zambia: A clarion call to scaling-up “One Health” research in the wake of emerging and re-emerging infectious diseases" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Dear Authors,

Thank you for submitting of your manuscript to PLOSNTD. I regret this long evaluation time – multiple experts had been invited before three finally agreed and now sent their reviews (please see below).

Your methodology was cleary articulated however one of our reviewers suggested to add more criteria. Regardings to your results, Table 1 and 2 should be reformatted in order to clearly show the lack of information available on pathogen detection. Please rewrite your results and separate the discussion part. In addition, the authors should provide more information such as cross-reactivity of antibodies between flaviviruses in serological surveys, discussion on vector control efforts and policy to combat zoonoses in Zambia. The opposite results of two studies should be add more discussion. Finally, the limitation of the study should be clearly described.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

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Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Sincerely,

Sirikachorn Tangkawattana, Ph.D.

Associate Editor

PLOS Neglected Tropical Diseases

Paulo Pimenta

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: I have no methodological concerns. The authors execute an appropriately designed scoping review to achieve the stated objective of identifying research gaps for vector-borne zoonotic pathogens in Zambia.

Reviewer #2: As this was a scoping review article, many of the methodology criteria do not apply. However, in areas where the methods are outlined (lines 150-164), it would be nice to see how many articles were identified, and then which exclusionary filters were imposed. Including the number of studies that were removed at each step would also be ideal. For instance, "articles in languages other than English, n = __". Doing so would also provide more evidence that VBD are understudied in Zambia.

Reviewer #3: -The objectives of the study was clearly articulated a with the study design appropriately addressing the stated objectives.

Adequate literature was reviewed to make up sufficient sample size to ensure adequate power to address the hypothesis being tested.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Lines 250-254: “Recently, a study conducted in 2016 among residents of Lukanga swamps in Central Province, revealed a 36.9% (79/214) sero-positivity for CHIKV and 19.6% (42/214) for Mayaro virus (MAYV) [24]. A recent study that screened for alphaviruses using 9,699 mosquitoes collected in Zambia (2014-2017) could not detect known alphaviruses (CHIKV, O'nyong'nyong virus, SINV etc.) [37].” Taken together, these results are quite striking – the two studies report essentially opposite findings. The authors should provide additional commentary on the research gap that is highlighted by these contrasting results, and propose hypotheses as to why such high seropositivity is observed near Lukanga, while the collection of nearly 10,000 mosquitos in Zambia resulted in no alphavirus detection.

Reviewer #2: Would recommend reformatting of Tables 1 & 2, which I outline in more detail within my general comments. Ultimately, generating some form of table or figure clearly showing how many studies were detected for each category (serologic vs. pathogen detection) and either stratifying on the basis of vector (as they have done) or pathogen type (virus, bacteria, parasite, etc) would be helpful. This would also highlight the lack of information available on pathogen detection as compared to serologic detection.

Reviewer #3: The analysis presented match the analysis plan however a substantial part of the " Results" as presented was suppose to have been captured under "Discussion".

From line 422 to the end should have been recommendation not results.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Lines 427-429: The authors correctly highlight a weakness of the existing literature for Zambia, that “the majority of the studies were serological in nature.” Particularly because of their focus on arboviruses, the authors should highlight the extensive and well-documented cross-reactivity of antibodies between flaviviruses of different species. This represents a major caveat for interpreting any epidemiological studies that rely solely on flaviviral serosurvey data. The poor specificity of flaviviral serologic tests makes it quite challenging to derive accurate estimates of disease attributable to specific flaviviral species. Highlighting this methodological weakness of serosurveys further underscores the authors’ call “for increased genomic surveillance” (Line 430) as a complementary experimental approach.

In the present manuscript, there is no discussion of vector control efforts to combat zoonoses in Zambia, however understanding Zambia’s history of vector control is necessary to contextualize the reviewed studies. To this end, the authors should include a brief discussion of major vector control policies and efforts/campaigns (insecticide spraying, etc.) that have taken place in Zambia and focused on zoonotic pathogens, particularly if there are studies that measured vector populations or disease incidence pre- and post- control interventions. (Even if there are no published studies, such data may be available from the Ministry of Health.) If there are no such studies, the authors should explicitly highlight this as a research gap.

Reviewer #2: Yes, though as I outline in other parts of my review, I would like to see Tables 1 & 2 modified to highlight at higher resolution where the knowledge gaps lie, and perhaps use that more granular analysis to present some detailed/specific multi-pronged VBD surveillance strategies. Including some preliminary policy recommendations, or at least identifying funding priorities, would be helpful to begin actualizing or making tangible some of the actions that could be taken to enhance our understanding of VBD dynamics in Zambia.

Reviewer #3: The study did not caption "conclusions" notwithstanding some of the findings of the study were supported by the data presented while some were not. Example of the not part of the study: The conclusion that the sparse capacity for diagnosis of vector borne pathogens is as a result of Zambian healthcare facilities being very limited, if any exist at all.

The limitations of analysis were not clearly described

The authors discuss how these data can be helpful to advance our understanding of the topic under study with public health relevance addressed

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: None.

Reviewer #2: Recommend reformatting Tables 1 & 2; please see Summary & General Comments. Are all papers that were reviewed cited somewhere in the paper? Rather, is it possible to find all papers covered within this scoping review within the references list?

Reviewer #3: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This manuscript by Mubemba et al. is a scoping review of vector-borne zoonotic pathogens in Zambia, with a focus on pathogens transmitted by mosquitos (particularly arboviruses), ticks, fleas, and tsetse flies. Overall, this is a strong, focused manuscript that achieves its stated objective of highlighting research gaps for vector-borne zoonoses in Zambia. The review also highlights some interesting emerging research areas specific to Zambia, particularly Q fever, Yersinia pestis, and multiple pathogens that were discovered in Zambia. As such, this manuscript provides an important foundation for setting research and public health priorities for these pathogens in Zambia and in sub-Saharan Africa more broadly. There are some minor typographical and grammatical errors that will need to be corrected before final publication, however overall the manuscript covers a diverse array of studies while summarizing them clearly and succinctly. My critiques are minor and focus on areas of the Discussion that should be expanded upon by the authors to emphasize research gaps, in support their main thesis.

I recommend Minor Revisions before the manuscript is accepted for publication.

Reviewer #2: This scoping review examined current knowledge and identified research gaps of vector-borne zoonotic pathogens in Zambia. The authors used several major scientific databases and a selected set of search terms to identify the presence of articles on this topic. Major findings included that there was a lack of representation of articles describing infection by means of pathogen isolation or detection of nucleic acid. Conversely, most articles to date have provided information on serological evidence of infection. Serological evidence is useful for identifying past exposure, but these data are limited in their interpretation. The authors have made good note of this, as they state that human burden of disease is difficult to attribute to any of these pathogens when the only information we have on host-pathogen interaction is seroconversion/evidence of exposure. Knowledge gaps in this area make implementation of national policy and advocating for transmission control funding difficult.

The authors have compiled a compelling body of work, highlighting the major knowledge gaps surrounding vector-borne disease circulation in Zambia. I really enjoyed reading this piece and agree with the authors on many points. A few areas worth building out/commenting on:

Related to flaviviruses, there is a high degree of serologic cross-reactivity, so by pinpointing that the majority of what we know about mosquito-borne flavivirus circulation in Zambia is based on serologic evidence is concerning for many reasons - one being that in general, flavivirus serology is not very reliable (for many cases, and this is dependent upon the diagnostic platform being used).

I would recommend re-formatting Table 1 to highlight which of the studies indicate serologic evidence of infection and which describe genomic detection. You might consider having a darkened circle for genomic detection, open circle for serologic, and half-filled circle for both. You could even list the reference numbers in the box alongside the circle, which would allow for easier navigation to those articles of interest by the reader, and also eliminate the fifth column in the table.

I appreciate the suggestions for increasing genomic surveillance. This portion of the discussion could be built out a bit more with specific suggestions for how to do this in a cost-effective manner. For instance, using banked samples (for instance, does Zambia National Blood Transfusion Service (ZNBTS) have banked sera?) and consensus screening tools (pan-flavivirus, for instance) to maximize detection rates. Further, you might recommend increasing genomic surveillance not only in vectors and reservoir hosts, but in companion animals and domestic livestock that share habitat and interact frequently with humans - essentially, using them as sentinels for arboviral circulation in human populations. While there are limitations to this approach with regard to arthropod bloodmeal/host preference, this would allow for an analysis into which pathogens are being transmitted by arthropods in the same geographic region.

I would also build out Table 2 in a way that stratifies the suggestions. You could stratify each row by capacity-building, diagnostics, and surveillance… and then make recommendations for priority funding areas if that is possible. You might also consider stratifying each row on the basis of geographic regions of coverage… so for instance, “hospital/focal surveillance”, “regional surveillance”, or more integrated national surveillance programs to track pathogen spread and evolution. Another stratification could be “immediate”, “short-term”, and “long-term”, where more immediate recommendations might include some of the education/awareness campaigns you outline in the discussion. These are just a few ideas, and I suspect you could do this in a number of different ways to help drive home the impact of these recommendations. Table 2 is really the big take-home from this paper and could be featured in a way that allows for strong identification of patterns and then building off of that, some solid policy recommendations, or at least identification of funding priorities.

Reviewer #3: -Why was the focus only on mosquitoes, ticks, fleas and tsetsefly as vectors and not others such as blackfly etc

- Line 126 to 129 should be referenced otherwise it is a serious assertion to make if unsubstantiated

-On line 191, it is stated that Culex mosquito is a "suspected " but not a confirmed vector of NTAV. Which is the confirmed one then

-Which other countries have been reported for NTAV, dengue, zika, WNV, Chikungunya, RVF, tick borne bacterial diseases and flea borne zoonotic pathogens as reported for yellow fever which is in 32 other tropical countries aside Zambia

-When was the first case of encephalitis, zika and CCHF recorded in Zambia

-Are there no mosquito borne bacteria infections' study in Zambia?

-Which of the infection in line 274 and 275 is line 276-277 describing its symptoms?

-What are the symptoms of borreliosis and Q-fever

-Edit the name of the Table 1 by removing the first words which is 'A summary table showing the number of' and add of Zambia after the 'humans'

- A footer should be shown below Table 1 to show what ' X ' signifies

-Give reference to the sentence on line 465

- On line 468, it is stated that '...should be targeted for both field and facility-based surveillance efforts' – remove the word 'effort'

- Give reference to the sentences in lines 474 to 476

- On line 576-578, the sentence is speculative and should be stated as such

- Acknowledgments on line 586 is spelt wrongly

-The 'results' section had a lot of discussion which should be moved to the 'discussion' section. Rewrite your results and discussion.

--------------------

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13 Dec 2021

Submitted filename: Response_to_Reviewers.docx

Click here for additional data file.

18 Jan 2022

Dear Dr Simulundu,

Thank you very much for submitting your manuscript "Current knowledge of vector-borne zoonotic pathogens in Zambia: A clarion call to scaling-up “One Health” research in the wake of emerging and re-emerging infectious diseases" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Sirikachorn Tangkawattana, Ph.D.

Associate Editor

PLOS Neglected Tropical Diseases

Paulo Pimenta

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: No concerns.

Reviewer #2: Table 1 is much improved with the stratification of publications based on whether detection was serologic or molecular in nature.

Reviewer #3: (No Response)

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: No concerns.

Reviewer #2: It is striking to me how unevenly distributed Table 1 is. Look at the number of papers providing evidence of exposure to mosquito-borne arboviruses in humans compared to parallel data available on animals and vectors. It appears that surveillance of mosquito-borne viruses in vectors and animals is hugely lacking -- and those data are critical when considering these transmission cycles through a One Health lens. Conversely, there are MANY papers demonstrating molecular detection of bacterial/protozoal tick-borne, flea-borne, and tsetse-borne diseases in animals and arthropod vectors, and to a lesser extent in humans.

Reviewer #3: (No Response)

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: No concerns.

Reviewer #2: I think discussing how targeting only one aspect of the One Health triad (or in this case, one aspect of the transmission cycle) risks over-interpretation of the role that component plays in the cycle, and the general imbalance between studies suggests that this pattern may translate to our overall understanding of these processes on a larger scale. I see now that you’ve included in the beginning of the conclusions that it’s problematic that a majority of human-focused studies contained only serologic evidence, complicating the interpretation of this data (or ability to associate it with disease burden, levels of shedding/viremia, etc.) However, I think it’s worth elaborating on the point you make in lines 146-149. WHY is there such an imbalance in molecular detections? Sampling bias?

Reviewer #3: (No Response)

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: No concerns.

Reviewer #2: Line items:

• On page 19 of document, lines 419-422, it appears something happened with the ‘track changes’, as I believe the last sentence of that section is fragmented.

• Missing reference in page 2 of the discussion, and page 6 (line 118, 121) of discussion,

• Line 123: ‘one-health’ is used but in the rest of the paper, you use One-Health (capitalized). Also, I think I’ve seen it used more often in the past without a hyphen, but I’ll defer to the editor on that.

• There were some comments in the conclusions (lines 144-145) that I found a bit confusing.

Reviewer #3: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This manuscript by Mubemba et al. is a scoping review of vector-borne zoonotic pathogens in Zambia, with a focus on pathogens transmitted by mosquitos (particularly arboviruses), ticks, fleas, and tsetse flies. This work provides an important foundation for setting research and public health priorities for these pathogens in Zambia and in sub-Saharan Africa more broadly. While there remain some minor typographical and grammatical errors that will require editing before final publication, in terms of content, the authors have fully addressed all my critiques. Indeed, the authors' revisions to Table 1 (based on the suggestions of Reviewer #2) have markedly improved the clarity and visual presentation of the literature review. I therefore support acceptance of the manuscript for publication.

Reviewer #2: (No Response)

Reviewer #3: All scientific names should be italized

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Reviewer #1: Yes: Joshua R. Lacsina

Reviewer #2: Yes: Anna C Fagre

Reviewer #3: No

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References

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20 Jan 2022

Submitted filename: Letter_Response_to_Reviewers.docx

Click here for additional data file.

24 Jan 2022

Dear Dr Simulundu,

We are pleased to inform you that your manuscript 'Current knowledge of vector-borne zoonotic pathogens in Zambia: A clarion call to scaling-up “One Health” research in the wake of emerging and re-emerging infectious diseases' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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Associate Editor

PLOS Neglected Tropical Diseases

Paulo Pimenta

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

28 Jan 2022

Dear Dr Simulundu,

We are delighted to inform you that your manuscript, "Current knowledge of vector-borne zoonotic pathogens in Zambia: A clarion call to scaling-up “One Health” research in the wake of emerging and re-emerging infectious diseases," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases