Molecular identification of Crimean-Congo haemorrhagic fever virus in Hyalomma rufipes and Amblyomma variegatum in the Upper East Region of Ghana.

Sampled ticks were screened for Crimean-Congo haemorrhagic fever virus (CCHFV) using an assay that targets the nucleoprotein gene region of the S segment, a conserved region of the CCHFV genome. Minimum infection rates of 0.34% and 0.10% were obtained when testing pools of Hyalomma rufipes and Amblyomma variegatum, respectively. Next-generation sequencing and phylogenetic analysis showed that the S and L segments of the CCHFV isolate clustered with those of similar isolates of genotype III. However, analysis of the M segment showed that reassortment had occurred, causing this segment to cluster with those of isolates of genotype I, providing the first evidence of such an occurrence in Ghana.

First Molecular Identification of Rickettsia aeschlimannii and Rickettsia africae in Ticks from Ghana.

The threats from vector-borne pathogens transmitted by ticks place people (including deployed troops) at increased risk for infection, frequently contributing to undifferentiated febrile illness syndromes. Wild and domesticated animals are critical to the transmission cycle of many tick-borne diseases. Livestock can be infected by ticks, and serve as hosts to tick-borne diseases such as rickettsiosis. Thus, it is necessary to identify the tick species and determine their potential to transmit pathogens. A total of 1,493 adult ticks from three genera-Amblyomma, Hyalomma, and Rhipicephalus-were identified using available morphological keys and were pooled (n = 541) by sex and species. Rickettsia species were detected in 308 of 541 (56.9%) pools by genus-specific quantitative polymerase chain reaction assay (Rick17b). Furthermore, sequencing of the outer membrane protein A and B genes (ompA and ompB) of random samples of Rickettsia-positive samples led to the identification of Rickettsia aeschlimannii and Rickettsia africae with most R. africae DNA (80.2%) detected in pools of Amblyomma variegatum. We report the first molecular detection and identification of the rickettsial pathogens R. africae and R. aeschlimannii in ticks from Ghana. Our findings suggest there is a need to use control measures to prevent infections from occurring among human populations in endemic areas in Ghana. This study underscores the importance of determining which vector-borne pathogens are in circulation in Ghana. Further clinical and prevalence studies are needed to understand more comprehensively the clinical impact of these rickettsial pathogens contributing to human disease and morbidity in Ghana.

Spatial and Seasonal Patterns of Tick Infestations in Kassena-Nankana Livestock.

The ability of ticks to adapt to different ecological zones, coupled with the spread of infectious pathogens negatively affects livestock production and thus, there is a need for better control strategies. However, control measures within a geographical region can only be effective if there is available information on tick population dynamics and ecology. This study focused on ticks infesting livestock in the Kassena-Nankana Districts of the Upper East Region of Ghana. The ticks were morphologically identified, variables such as season, animal host, and predilection sites were recorded, and the data were analyzed using STATA version 13. Out of 448 livestock examined, tick infestation in cattle was (78.60%), followed by sheep (25%) and goats (5.88%). A total of 1,550 ticks including nymphs (303) and adults (1,247) were collected. Adult ticks were found to be significantly associated with season (p < 0.001), with a high burden in the wet season. The nymph burden and body parts of livestock hosts were significantly associated with more nymphs collected from male animals than females (p < 0.001). Three genera of ticks, Amblyomma (62.97%), Hyalomma (18.71%), and Rhipicephalus (18.32%) were morphologically identified with the most predominant tick species recorded as Amblyomma variegatum (62.97%). Matured A. variegatum was sampled primarily in the wet season with their predilection site as the udder/scrotum (p < 0.001). However, adult Hyalomma truncatum was observed to have a significant association with the anal region (p < 0.001). Findings from this study are essential for formulating tick control measures to prevent the spread of infectious pathogens.

First record of Babesia and Theileria parasites in ticks from Kassena-Nankana, Ghana.

Ticks are efficient vectors for transmitting pathogens that negatively affect livestock production and pose a risk to public health. In this study, Babesia and Theileria species were identified in ticks collected from cattle, sheep and goats from the Kassena-Nankana Districts of Ghana between February and December 2020. A total of 1550 ticks were collected, morphologically identified, pooled and screened for pathogens using primers that amplify a 560 bp fragment of the ssrRNA gene and Sanger sequencing. Amblyomma variegatum (62.98%) was the predominant tick species. From the 491 tick pools screened, 12/15 (2.44%) positive pools were successfully sequenced. The pathogen DNA identified were Theileria ovis in eight (15.38%) pools of Rhipicephalus evertsi evertsi, Theileria velifera in two (0.78%) pools of A. variegatum and Babesia occultans and Babesia sp. Xinjiang in one (1.72%) pool each of Hyalomma truncatum. It was further observed that T. ovis occurred in ticks collected from only sheep (p < 0.001) which were females (p = 0.023) and < =1 year old (p = 0.040). This study reports the first identification of these pathogens in ticks within Kassena-Nankana. With the constant trade of livestock, there is a need for effective tick control measures to prevent infection spread.

Molecular survey of Anaplasma and Ehrlichia species in livestock ticks from Kassena-Nankana, Ghana; with a first report of Anaplasma capra and Ehrlichia minasensis.

Tick-borne pathogens harm livestock production and pose a significant risk to public health. To combat these effects, it is necessary to identify the circulating pathogens to create effective control measures. This study identified Anaplasma and Ehrlichia species in ticks collected from livestock in the Kassena-Nankana Districts between February 2020 and December 2020. A total of 1550 ticks were collected from cattle, sheep and goats. The ticks were morphologically identified, pooled and screened for pathogens using primers that amplify a 345 bp fragment of the 16SrRNA gene and Sanger sequencing. The predominant tick species collected was Amblyomma variegatum (62.98%). From the 491 tick pools screened, 34 (6.92%) were positive for Ehrlichia and Anaplasma. The pathogens identified were Ehrlichia canis (4.28%), Ehrlichia minasensis (1.63%), Anaplasma capra (0.81%) and Anaplasma marginale (0.20%). This study reports the first molecular identification of the above-mentioned Ehrlichia and Anaplasma species in ticks from Ghana. With the association of human infections with the zoonotic pathogen A. capra, livestock owners are at risk of infections, calling for the development of effective control measures.

Ticks and prevalence of tick-borne pathogens from domestic animals in Ghana.

BACKGROUND: Ticks are important vectors of various pathogenic protozoa, bacteria and viruses that cause serious and life-threatening illnesses in humans and animals worldwide. Estimating tick-borne pathogen prevalence in tick populations is necessary to delineate how geographical differences, environmental variability and host factors influence pathogen prevalence and transmission. This study identified ticks and tick-borne pathogens in samples collected from June 2016 to December 2017 at seven sites within the Coastal, Sudan and Guinea savanna ecological zones of Ghana. METHODS: A total of 2016 ticks were collected from domestic animals including cattle, goats and dogs. Ticks were morphologically identified and analysed for pathogens such as Crimean-Congo haemorrhagic fever virus (CCHFV), Alkhurma haemorrhagic fever virus (AHFV), Rickettsia spp. and Coxiella burnetii using polymerase chain reaction assays (PCR) and sequence analysis. RESULTS: Seven species were identified, with Amblyomma variegatum (60%) most frequently found, followed by Rhipicephalus sanguineus sensu lato (21%), Rhipicephalus spp. (9%), Hyalomma truncatum (6%), Hyalomma rufipes (3%), Rhipicephalus evertsi (1%) and Rhipicephalus (Boophilus) sp. (0.1%). Out of 912 pools of ticks tested, Rickettsia spp. and Coxiella burnetii DNA was found in 45.6% and 16.7% of pools, respectively, whereas no CCHFV or AHFV RNA were detected. Co-infection of bacterial DNA was identified in 9.6% of tick pools, with no statistical difference among the ecozones studied. CONCLUSIONS: Based on these data, humans and animals in these ecological zones are likely at the highest risk of exposure to rickettsiosis, since ticks infected with Rickettsia spp. displayed the highest rates of infection and co-infection with C. burnetii, compared to other tick-borne pathogens in Ghana.

Deciphering pyrethroid resistance in Cx. pipiens (L): Implications of cytochrome P450; expression profiling and regulatory microRNA.

Over the past decades, the extensive use of pyrethroids insecticides for vector control has resulted in the development of insecticide resistance. Cytochrome P450 has been recognized to play a critical role in the metabolic detoxification of insecticides. In the current study, Culex pipiens mosquitoes were collected from Giza Governorate in Egypt and tested for insecticide susceptibility against deltamethrin. First detection of Knockdown resistance gene (Kdr) mutations in field collected mosquitoes was performed. Activities of cytochrome oxidase P450 detoxification enzyme that synchronized with the resistance development, was assessed. Expression profiles of cytochrome P450s and their putative corresponding regulating miRNAs, which was previously reported in Cx. pipiens pallens were evaluated in pyrethroid resistant field-collected Cx. pipiens using RT-qPCR and stem-loop RT-qPCR, respectively. Specific stem-loop reverse transcription primers and forward primers were designed for miRNAs profiling. Our results elucidated the pyrethroid resistance development and revealed its relation to the metabolic and target site modification mechanisms with a first report of L1014F-kdr mutation detection. RT-qPCR results have showed an up-regulation in the expression of the studied P450 transcripts. Negative correlations were found between the expression of P450s and their regulatory miRNAs except for CYP9J35, where positive correlation was found with its corresponding miR-13. Interestingly, our data was the first to detect negative correlation between miR-285 and its putative CYP6Cp1 target gene. These findings highlighted the significance of identifying P450 gene along with regulatory miRNAs as a key mechanism implicated in pyrethroid resistance in field Culex vector population. The elucidation of this mechanism would shed light on the development of insecticide resistance and would help in shaping strategies to combat such vectors.

Preliminary Screening of Mosquito Spatial Distribution in Togo: With Special Focus on the Aedes (Diptera: Culicidae) Species.

The Togolese Republic has a tropical and humid climate which constitutes an ideal environment for mosquitoes to breed and transmit diseases. The Aedes mosquito is known to transmit yellow fever (YF), dengue, chikungunya, and Zika viruses in West Africa. Togo has been suffering from YF virus transmission, despite vaccination efforts. Unfortunately, there is scarcity in the data that reflect mosquito spatial distribution in Togo, specifically possible YF vectors. In the current study, mosquito surveillance efforts targeted areas with confirmed YF cases between July and August 2012. Indoor mosquitoes were collected using knockdown insecticide spraying, whereas Biogents (BG) traps were used to collect outdoor mosquito adults. Mosquito larval surveillance was conducted as well. In total, 17 species were identified. This investigation revealed the presence of medically important vectors in Togo, especially the Aedes aegypti (Linnaeus) (Diptera: Culicidae) which was collected in the four regions. Screening of all pools of female Aedes mosquitoes for YF, by real-time PCR, showed negative results. This is the first record for Coquillettidia flavocincta (Edwards) (Diptera: Culicidae) species in West Africa. This preliminary work serves as a baseline for further mosquito distribution studies in Togo.

Xenosurveillance reflects traditional sampling techniques for the identification of human pathogens: A comparative study in West Africa.

BACKGROUND: Novel surveillance strategies are needed to detect the rapid and continuous emergence of infectious disease agents. Ideally, new sampling strategies should be simple to implement, technologically uncomplicated, and applicable to areas where emergence events are known to occur. To this end, xenosurveillance is a technique that makes use of blood collected by hematophagous arthropods to monitor and identify vertebrate pathogens. Mosquitoes are largely ubiquitous animals that often exist in sizable populations. As well, many domestic or peridomestic species of mosquitoes will preferentially take blood-meals from humans, making them a unique and largely untapped reservoir to collect human blood. METHODOLOGY/PRINCIPAL FINDINGS: We sought to take advantage of this phenomenon by systematically collecting blood-fed mosquitoes during a field trail in Northern Liberia to determine whether pathogen sequences from blood engorged mosquitoes accurately mirror those obtained directly from humans. Specifically, blood was collected from humans via finger-stick and by aspirating bloodfed mosquitoes from the inside of houses. Shotgun metagenomic sequencing of RNA and DNA derived from these specimens was performed to detect pathogen sequences. Samples obtained from xenosurveillance and from finger-stick blood collection produced a similar number and quality of reads aligning to two human viruses, GB virus C and hepatitis B virus. CONCLUSIONS/SIGNIFICANCE: This study represents the first systematic comparison between xenosurveillance and more traditional sampling methodologies, while also demonstrating the viability of xenosurveillance as a tool to sample human blood for circulating pathogens.

Virus genomes reveal factors that spread and sustained the Ebola epidemic.

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics.

West African Anopheles gambiae mosquitoes harbor a taxonomically diverse virome including new insect-specific flaviviruses, mononegaviruses, and totiviruses.

Anopheles gambiae are a major vector of malaria in sub-Saharan Africa. Viruses that naturally infect these mosquitoes may impact their physiology and ability to transmit pathogens. We therefore used metagenomics sequencing to search for viruses in adult Anopheles mosquitoes collected from Liberia, Senegal, and Burkina Faso. We identified a number of virus and virus-like sequences from mosquito midgut contents, including 14 coding-complete genome segments and 26 partial sequences. The coding-complete sequences define new viruses in the order Mononegavirales, and the families Flaviviridae, and Totiviridae. The identification of a flavivirus infecting Anopheles mosquitoes broadens our understanding of the evolution and host range of this virus family. This study increases our understanding of virus diversity in general, begins to define the virome of a medically important vector in its natural setting, and lays groundwork for future studies examining the potential impact of these viruses on anopheles biology and disease transmission.

Reduced evolutionary rate in reemerged Ebola virus transmission chains.

On 29 June 2015, Liberia's respite from Ebola virus disease (EVD) was interrupted for the second time by a renewed outbreak ("flare-up") of seven confirmed cases. We demonstrate that, similar to the March 2015 flare-up associated with sexual transmission, this new flare-up was a reemergence of a Liberian transmission chain originating from a persistently infected source rather than a reintroduction from a reservoir or a neighboring country with active transmission. Although distinct, Ebola virus (EBOV) genomes from both flare-ups exhibit significantly low genetic divergence, indicating a reduced rate of EBOV evolution during persistent infection. Using this rate of change as a signature, we identified two additional EVD clusters that possibly arose from persistently infected sources. These findings highlight the risk of EVD flare-ups even after an outbreak is declared over.

Insecticide susceptibility of natural populations of Anopheles coluzzii and Anopheles gambiae (sensu stricto) from Okyereko irrigation site, Ghana, West Africa.

BACKGROUND: The increasing spread of insecticide resistance in malaria vectors has been well documented across sub-Saharan Africa countries. The influence of irrigation on increasing vector resistance is poorly understood, and is critical to successful and ethical implementation of food security policies. This study investigated the insecticide resistance status of An. gambiae (s.l.) mosquitoes collected from the irrigated rice area of Okyereko, a village containing about 42 hectares of irrigated field within an irrigation project plan in the Central Region of Ghana. Large amounts of insecticides, herbicides and fertilizers are commonly used in the area to boost the annual production of the rice. METHODS: Mosquito larvae were collected and adults were assayed from the F1 progeny. The resistance status, allele and genotype were characterized using WHO susceptibility testing and PCR methods respectively. RESULTS: The An. gambiae (s.l.) populations from Okyereko are highly resistant to DDT and pyrethroid insecticides, with possible involvement of metabolic mechanisms including the elevation of P450 and GST enzyme as well as P-gp activity. The population was mostly composed of An. coluzzii specimens (more than 96 %) with kdr and ace-1 frequencies of 0.9 and 0.2 %, respectively. CONCLUSION: This study brings additional information on insecticide resistance and the characterization of An. gambiae (s.l.) mosquitoes from Okyereko, which can be helpful in decision making for vector control programmes in the region.

Evolution and Spread of Ebola Virus in Liberia, 2014-2015.

The 2013-present Western African Ebola virus disease (EVD) outbreak is the largest ever recorded with >28,000 reported cases. Ebola virus (EBOV) genome sequencing has played an important role throughout this outbreak; however, relatively few sequences have been determined from patients in Liberia, the second worst-affected country. Here, we report 140 EBOV genome sequences from the second wave of the Liberian outbreak and analyze them in combination with 782 previously published sequences from throughout the Western African outbreak. While multiple early introductions of EBOV to Liberia are evident, the majority of Liberian EVD cases are consistent with a single introduction, followed by spread and diversification within the country. Movement of the virus within Liberia was widespread, and reintroductions from Liberia served as an important source for the continuation of the already ongoing EVD outbreak in Guinea. Overall, little evidence was found for incremental adaptation of EBOV to the human host.

Comparison of Indoor Residual Spray Equipment for Malaria Control in Liberia.

We describe and compare a new innovative backpack compressed-air sprayer (JQSX-12) to a Stihl® 450 backpack mist blower and a manually operated compression sprayer for its effectiveness as an alternative operational tool for indoor residual insecticide application to control malaria in Liberia. Advantages and physical characteristics of each sprayer and their spray atomization parameters are discussed.

Molecular Evidence of Sexual Transmission of Ebola Virus.

A suspected case of sexual transmission from a male survivor of Ebola virus disease (EVD) to his female partner (the patient in this report) occurred in Liberia in March 2015. Ebola virus (EBOV) genomes assembled from blood samples from the patient and a semen sample from the survivor were consistent with direct transmission. The genomes shared three substitutions that were absent from all other Western African EBOV sequences and that were distinct from the last documented transmission chain in Liberia before this case. Combined with epidemiologic data, the genomic analysis provides evidence of sexual transmission of EBOV and evidence of the persistence of infective EBOV in semen for 179 days or more after the onset of EVD. (Funded by the Defense Threat Reduction Agency and others.).

Monitoring of Ebola Virus Makona Evolution through Establishment of Advanced Genomic Capability in Liberia.

To support Liberia's response to the ongoing Ebola virus (EBOV) disease epidemic in Western Africa, we established in-country advanced genomic capabilities to monitor EBOV evolution. Twenty-five EBOV genomes were sequenced at the Liberian Institute for Biomedical Research, which provided an in-depth view of EBOV diversity in Liberia during September 2014-February 2015. These sequences were consistent with a single virus introduction to Liberia; however, shared ancestry with isolates from Mali indicated at least 1 additional instance of movement into or out of Liberia. The pace of change is generally consistent with previous estimates of mutation rate. We observed 23 nonsynonymous mutations and 1 nonsense mutation. Six of these changes are within known binding sites for sequence-based EBOV medical countermeasures; however, the diagnostic and therapeutic impact of EBOV evolution within Liberia appears to be low.

Sampling host-seeking anthropophilic mosquito vectors in west Africa: comparisons of an active human-baited tent-trap against gold standard methods.

In this study, we characterize the ability of the previously described Infoscitex tent (IST) to capture mosquitoes in comparison to either the Centers for Disease Control Light Trap hung next to individuals under a bed net (LTC) or to human landing catches (HLC). In Senegal, the IST caught 6.14 times the number of Anopheles gambiae sensu lato (s.l.), and 8.78 times the Culex group V mosquitoes as LTC. In one of two locations in Burkina Faso, the IST caught An. gambiae at a rate not significantly different than HLC. Of importance, 9.1-36.1% of HLC caught An. gambiae were blood fed, mostly with fresh blood, suggesting they fed upon the collector, whereas only 0.5-5.0% from the IST had partial or old blood. The IST also caught outdoor biting species in proportions comparable to HLC. The results show this tent provides a safer and effective alternative to the skill-dependent, risky, and laborious HLC method.

Swarming mechanisms in the yellow fever mosquito: aggregation pheromones are involved in the mating behavior of Aedes aegypti.

Mosquitoes of various species mate in swarms comprised of tens of thousands of flying males. In this study, we examined Aedes aegypti swarming behavior and identified associated chemical cues. Novel evidence is provided that Ae. aegypti females aggregate by means of olfactory cues, such as aggregation pheromones. Isolation of Ae. aegypti aggregation pheromones was achieved by aeration of confined mosquitoes and collection of associated volatiles by glass filters. The collected volatiles were identified through gas chromatography mass spectrometry (GCMS). Three aggregation pheromones were collected and identified as 2,6,6-trimethylcyclohex-2-ene-1,4-dione (ketoisophorone) (CAS# 1125-21-9, t(R) = 18.75), 2,2,6-trimethylcyclohexane-1,4-dione (the saturated analog of ketoisophorone) (CAS# 20547-99-3, t(R) = 20.05), and 1-(4-ethylphenyl) ethanone (CAS# 937-30-4, t(R) = 24.22). Our biological studies revealed that the identified compounds stimulated mosquito behavior under laboratory conditions. The mechanism of mosquito swarm formation is discussed in light of our behavioral study findings. A preliminary field trial demonstrated the potential application of the isolated aggregation pheromones in controlling Ae. aegypti.