A validated triplex RT-qPCR protocol to simultaneously detect chikungunya, dengue and Zika viruses in mosquitoes.

BACKGROUND & OBJECTIVES: Recently, the incidences of chikungunya, dengue and Zika infections have increased due to globalization and urbanization. It is vital that reliable detection tools become available to assess the viral prevalence within mosquito populations. METHODS: Based on the previous publications on clinical diagnosis in human infections, for the first time, we described a customized triplex RT-qPCR protocol for simultaneous detection of chikungunya virus (CHIKV), dengue virus serotypes 1-4 (DENV1-4) and Zika virus (ZIKV) in mosquitoes. RESULTS: In preliminary assessment to determine the specificity and sensitivity of primers and probes, all six targets were detected individually with the following thresholds as indicated by calculated pfu equivalents: 3.96x100 for CHIKV, 3.80x101 for DENV1, 3.20x101 for DENV2, 8.00x104 for DENV3, 1.58x100 for DENV4, and 6.20x100 for ZIKV When tested in a full combination of six targets (CDZ mix), CHIKV, DENV1-4 mix or ZIKV were all detected with the thresholds of 1.32x100 for CHIKV, 3.79x100 for DENV1-4 and 2.06x100 for ZIKV All targets, individually or in full combination were detected in the mixtures of Aedes aegypti (L.) homogenate and viral lysates. A robust evaluation with three replicates in each of three plates for CHIKV, DENV1-4 and ZIKV individually or in full combination was conducted. In individual assays, CHIKV was detected to 3.96x10-1, DENV1-4 to 1.14x100 and ZIKV to 3.20x100. In full combination assays, CHIKV was detected to 1.32x104, DENV1-4 to 3.79x101 and ZIKV to 1.07x100. INTERPRETATION & CONCLUSION: This triplex RT-qPCR assay appears to consistently detect all six targets and does not cross react with Ae. aegypti homogenate, making it a feasible, practical, and immediately adoptable protocol for use among vector control and other entities, particularly in the endemic areas of CHIKV, DENVs and ZIKV.

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.

Incursion and establishment of the Old World arbovirus vector Aedes (Fredwardsius) vittatus (Bigot, 1861) in the Americas.

Routine biosurveillance efforts at the Naval Station Guantanamo Bay, Cuba, on 18 June 2019, detected two unusual mosquitos in a CO2-baited CDC light trap. Morphological and molecular analysis confirmed the presence of Aedes (Fredwardsius) vittatus (Bigot, 1861) - the first record of the Old World dengue, chikungunya, Zika and yellow fever virus vector into the Americas - and provides evidence for its establishment in Cuba. Newly submitted GenBank sequences from Dominican Republic further evidence its establishment in the Caribbean, and a median-joining network analysis using mitochondrial COI gene sequences clearly supports multiple introductions of Ae. vittatus into the Caribbean from the Indian subcontinent. It was determined that many Ae. vittatus COI barcode sequences in GenBank are currently misidentified as Aedes (Fredwardsius) cogilli Edwards, 1922.

Susceptibility Profile of Aedes aegypti L. (Diptera: Culicidae) from Montclair, California, to Commonly Used Pesticides, With Note on Resistance to Pyriproxyfen.

The peridomestic anthropophilic Aedes aegypti L. (Diptera: Culicidae) is originated from the wild zoophilic subspecies Aedes aegypti formosus in sub-Saharan Africa, and currently has a broad distribution in human-modified environments of the tropics and subtropics worldwide. In California, breeding populations were initially detected in 2013 in the cities of Fresno, Madera, and San Mateo, and now can be found in 188 cities of 12 counties in the state. Recent genetic studies suggest that this species invaded California on multiple occasions from several regions of the United States and northern Mexico prior to initial detection. As an invasive species and vector for numerous arboviruses, Ae. aegypti is a primary target of surveillance and control in California. In southern California city of Montclair, a population was identified in September 2015, from which a short-term colony was established in an insectary. The susceptibility of this field population to commonly used pesticides with various modes of action, including 15 formulations against larvae and four against adults, was determined, in reference to a susceptible laboratory colony of the same species. No resistance was shown to most pesticides tested. However, tolerance or reduced susceptibility to spinosad, spinetoram, diflubezuron, and fipronil was detected, and modest levels of resistance to pyriproxyfen (resistance ratio = 38.7-fold at IE50 and 81.5-fold at IE90) was observed. Results are discussed based on the field usage and modes of action of the pesticides tested. Strategic selection and application of pesticides against this population of Ae. aegypti in the urban environments should be taken into consideration.

High Resistance to Bacillus sphaericus and Susceptibility to Other Common Pesticides in Culex pipiens (Diptera: Culicidae) from Salt Lake City, UT.

Biorational mosquito larvicides based on microbial organisms and insect growth regulators (IGRs) have played a vital role in integrated mosquito control, particularly since the invasion of West Nile virus to the United States in 1999. Products that are formulated with technical powder of the bacterium, Bacillus sphaericus Neide (recently Lysinibacillus sphaericus Meyer and Neide), are among the ones that have been extensively applied to combat Culex and other mosquito species. Due to the simplicity of the binary toxins, resistance to this pesticide in laboratory and field populations of Culex pipiens L. complex has occurred globally since 1994. A Cx. pipiens population with a high level of resistance to B. sphaericus (VectoLex WDG) was identified in Salt Lake City, UT, in September 2016. The resistance ratios in this population were 20,780.0- and 23,926.9-fold at LC50 and LC90, respectively, when compared with a susceptible population of a laboratory reference colony of the same species. This B. sphaericus-resistant population remained mostly susceptible to other commonly used pesticides to control arthropods of public health and urban significance, including ones based on microbial organisms (Bacillus thuringiensis subsp. israelensis, spinosad, spinetoram, abamectin), IGRs (pyriproxyfen, methoprene, diflubenzuron, novaluron), organophosphate (temephos), neonicotinoid (imidacloprid), phenylpyrazole (fipronil), oxadiazine (indoxacarb), and pyrethroid (permethrin). Results are discussed according to the modes of action of the pesticides tested, and suggestions are made to manage B. sphaericus-resistant mosquito populations.

Host (avian) biting preference of southern California Culex mosquitoes (Diptera: Culicidae).

The host preference of a vector mosquito species plays a significant role in determining human and animal risk of infection with mosquito-transmitted pathogens. Host preferences of common southern California Culex species for four bird species, American crow (Corvus brachyrhynchos), house sparrow (Passer domesticus), house finch (Carpodacus mexicanus), and mourning dove (Zenaida macroura), were examined by determining the proportion of each mosquito species that successfully engorged on each of the four bird species presented equally within a net trap to wild host-seeking mosquitoes. Bloodmeals in engorged mosquitoes captured within the net trap were identified to avian species by using a multiplex polymerase chain reaction assay targeting the cytochrome b gene sequence. There were significant differences in host selection by all three Culex species captured in numbers sufficient for analysis, with Culex erythrothorax Dyar preferentially biting American crows, Culex tarsalis Coquillett preferentially biting house sparrows, and Culex quinquefasciatus Say preferentially biting house finches. All three Culex species demonstrated more frequent engorgement on passerine birds (sparrows, finches, and crows) than the nonpasserine mourning dove. A greater preference for passerine birds might be expected to increase the transmission of pathogens, such as West Nile virus, to which passerine birds are particularly competent hosts.