A Survey on Mosquito Control Knowledge and Insecticide Use in New Orleans, La, 2020-2021.

Mosquitoes are a known public nuisance and can vector various diseases. Historically, New Orleans, LA, has long been acquainted with the burden of mosquito-borne diseases, such as malaria and yellow fever in the 20th century and West Nile virus in the 21st century. Government mosquito control awareness campaigns have been around for decades as has the use of organophosphate and pyrethroid insecticides by mosquito abatement districts. However, few data are available on public perception of mosquito control and public usage of insecticides to kill mosquitoes in New Orleans. We conducted a survey from August 2020 to July 2021 to evaluate New Orleans residents' 1) general knowledge regarding mosquito control and 2) what measures and products they use to control mosquitoes. The aim of this survey was to determine how residents contribute to backyard mosquito control by do-it-yourself or professional applications of insecticides. The survey was disseminated both online and via mail. Of the 396 survey participants, nearly all (99.48%) agreed that mosquito control is important in New Orleans because it prevents mosquito bites (30.85%), prevents mosquito borne-diseases (38.51%), and prevents nuisance mosquitoes (29.17%). More than one-third (35%) of survey participants indicated that they empty containers to reduce adult mosquitoes on their own property. More than two-thirds of the participants (69.95%) would not hire a pest management professional to spray their yard for adult mosquitoes, and only 20% of survey participants do apply a pesticide to kill adult mosquitoes on their own property. None of our findings were associated with the level of education, gender, or age of participants. This study suggests that the City of New Orleans Mosquito, Termite and Rodent Control Board educational and outreach campaigns may be an effective tool in spreading mosquito control awareness and contribute to residents' knowledge of mosquito control. The data we collected indicate that residents understand what mosquito control is and why it is important in New Orleans.

A SURVEY ON MOSQUITO CONTROL KNOWLEDGE AND INSECTICIDE USE IN NEW ORLEANS, LA, 2020-2021.

Mosquitoes are a known public nuisance and can vector various diseases. Historically, New Orleans, LA, has long been acquainted with the burden of mosquito-borne diseases, such as malaria and yellow fever in the 20th century and West Nile virus in the 21st century. Government mosquito control awareness campaigns have been around for decades as has the use of organophosphate and pyrethroid insecticides by mosquito abatement districts. However, few data are available on public perception of mosquito control and public usage of insecticides to kill mosquitoes in New Orleans. We conducted a survey from August 2020 to July 2021 to evaluate New Orleans residents' 1) general knowledge regarding mosquito control and 2) what measures and products they use to control mosquitoes. The aim of this survey was to determine how residents contribute to backyard mosquito control by do-it-yourself or professional applications of insecticides. The survey was disseminated both online and via mail. Of the 396 survey participants, nearly all (99.48%) agreed that mosquito control is important in New Orleans because it prevents mosquito bites (30.85%), prevents mosquito borne-diseases (38.51%), and prevents nuisance mosquitoes (29.17%). More than one-third (35%) of survey participants indicated that they empty containers to reduce adult mosquitoes on their own property. More than two-thirds of the participants (69.95%) would not hire a pest management professional to spray their yard for adult mosquitoes, and only 20% of survey participants do apply a pesticide to kill adult mosquitoes on their own property. None of our findings were associated with the level of education, gender, or age of participants. This study suggests that the City of New Orleans Mosquito, Termite and Rodent Control Board educational and outreach campaigns may be an effective tool in spreading mosquito control awareness and contribute to residents' knowledge of mosquito control. The data we collected indicate that residents understand what mosquito control is and why it is important in New Orleans.

Genetic diversity of Trypanosoma cruzi infecting raccoons (Procyon lotor) in 2 metropolitan areas of southern Louisiana: implications for parasite transmission networks.

Trypanosoma cruzi, the aetiological agent of Chagas disease, exists as an anthropozoonosis in Louisiana. Raccoons are an important reservoir, as they demonstrate high prevalence and maintain high parasitaemia longer than other mammals. Given the complex nature of parasite transmission networks and importance of raccoons as reservoirs that move between sylvatic and domestic environments, detailing the genetic diversity of T. cruzi in raccoons is crucial to assess risk to human health. Using a next-generation sequencing approach targeting the mini-exon, parasite diversity was assessed in 2 metropolitan areas of Louisiana. Sequences were analysed along with those previously identified in other mammals and vectors to determine if any association exists between ecoregion and parasite diversity. Parasites were identified from discrete typing units (DTUs) TcI, TcII, TcIV, TcV and TcVI. DTUs TcII, TcV and TcVI are previously unreported in raccoons in the United States (US). TcI was the most abundant DTU, comprising nearly 80% of all sequences. All but 1 raccoon harboured multiple haplotypes, some demonstrating mixed infections of different DTUs. Furthermore, there is significant association between DTU distribution and level III ecoregion in Louisiana. Finally, while certain sequences were distributed across multiple tissues, others appeared to have tissue-specific tropism. Taken together, these findings indicate that ongoing surveillance of T. cruzi in the US should be undertaken across ecoregions to fully assess risk to human health. Given potential connections between parasite diversity and clinical outcomes, deep sequencing technologies are crucial and interventions targeting raccoons may prove useful in mitigating human health risk.

Raccoons As an Important Reservoir for Trypanosoma cruzi: A Prevalence Study from Two Metropolitan Areas in Louisiana.

Raccoons are an important reservoir for Trypanosoma cruzi infection, having been reported to maintain a high and lengthy parasitemia. Although raccoon populations have historically been abundant in Louisiana, the prevalence rate of T. cruzi infection in raccoons in this state is unknown. Here, we tested raccoon tissues from two urban areas in Louisiana, namely Orleans Parish (OP) and East Baton Rouge Parish (EBRP), to investigate prevalence in these areas using direct detection through polymerase chain reaction. Overall, 33.6% of raccoons tested were positive. The prevalence in OP (42.9%) was significantly higher than the prevalence in EBRP (23.2%). There was no significant difference in prevalence between sexes or based on age, but there was a significant difference in infection prevalence based on season of trapping. These results suggest the importance of raccoons as a reservoir host, maintaining T. cruzi infection and potentially posing a risk to human health.

Prevalence of Baylisascaris procyonis in Raccoons Trapped in New Orleans, Louisiana, 2014-2017.

Baylisascaris procyonis, the raccoon roundworm, is considered an emerging zoonotic disease in the United States after being identified in raccoons captured in different US regions and metropolitan areas. Humans can become infected with B. procyonis after ingesting mature roundworm eggs, sometimes resulting in fatal disease or severe sequelae in these patients. The first reported human Baylisascaris case in New Orleans raised concerns that very little was known about this zoonotic disease in the increasing urban raccoon population. Therefore, the study aim was to estimate the prevalence of Baylisascaris-infected raccoons in New Orleans, LA. Raccoons were trapped based on nuisance calls from the public and in areas where raccoons had been sighted. Necropsies were performed to identify B. procyonis adult worms in intestines and fecal samples were examined for roundworm ova. Study findings showed that 38.5% of New Orleans raccoons were infected with B. procyonis, defined by the presence of adult B. procyonis worms or Baylisascaris ova in their feces. Twenty-four of 65 raccoons (36.9%) had raccoon roundworms in their intestines and 31.7% of the fecal samples were positive for B. procyonis eggs. Mapping B. procyonis-infected raccoons by trapping location showed that infected raccoons were found throughout the city. In conclusion, the high prevalence rate of B. procyonis in New Orleans raccoons demonstrates the importance of educating the public and health care professionals about potential health risks and providing resources to prevent exposure to infective eggs from raccoon latrines. Furthermore, this emerging disease should be further studied to examine human risk of infection in increasing raccoon populations in metropolitan areas.

Field Trials to Evaluate the Effectiveness of the Biogents®-Sweetscent Lure in Combination with Several Commercial Mosquito Traps and to Assess the Effectiveness of the Biogents-Mosquitaire Trap with and Without Carbon Dioxide.

A series of field experiments was conducted in Florida, California, and Louisiana in order to investigate whether adding the Biogents® (BG)-Sweetscent lure to several commercially available mosquito traps increases their Aedes albopictus catch rates and to evaluate the BG-Mosquitaire trap with and without CO2. Adding the BG-Sweetscent to the SkeeterVac Bite-Guard SVE6211, MosClean UV LED (ultraviolet light-emitting diode), Flowtron® Galaxie PV 75, Dynatrap® DT2000XL, Bite Shield Protector, and Black Flag® BZ-40 increased their Ae. albopictus catch rates up to 4.2-fold. The catch rates of the BG-Mosquitaire and the BG-Sentinel did not differ significantly for Ae. aegypti and Culex quinquefasciatus. The BG-Mosquitaire without CO2 and only with BG-Sweetscent caught 1.2 times more Ae. albopictus than the CO2- and Lurex3-baited Mosquito Magnet® Patriot and 2.6 times more than the CO2- and Sweetscent-baited SkeeterVac, respectively. The BG-Mosquitaire baited with Sweetscent and CO2 collected 6.8 times more Ae. albopictus than the Mosquito Magnet Patriot and 11.9 times more than the SkeeterVac. We conclude that BG-Sweetscent increases the tiger mosquito catch rates of many commercially available mosquito traps. We proved that the BG-Mosquitaire is as efficient as the well-known BG-Sentinel and that it can outperform mosquito traps that are baited with propane-generated CO2.

Prevalence, distribution, and diversity of cryptic piroplasm infections in raccoons from selected areas of the United States and Canada.

The order Piroplasmida contains a diverse group of intracellular parasites, many of which can cause significant disease in humans, domestic animals, and wildlife. Two piroplasm species have been reported from raccoons (Procyon lotor), Babesia lotori (Babesia sensu stricto clade) and a species related to Babesia microti (called B. microti-like sp.). The goal of this study was to investigate prevalence, distribution, and diversity of Babesia in raccoons. We tested raccoons from selected regions in the United States and Canada for the presence of Babesia sensu stricto and Babesia microti-like sp. piroplasms. Infections of Babesia microti-like sp. were found in nearly all locations sampled, often with high prevalence, while Babesia sensu stricto infections had higher prevalence in the Southeastern United States (20-45% prevalence). Co-infections with both Babesia sp. were common. Sequencing of the partial 18S rRNA and cytochrome oxidase subunit 1 (cox1) genes led to the discovery of two new Babesia species, both found in several locations in the eastern and western United States. One novel Babesia sensu stricto sp. was most similar to Babesia gibsoni while the other Babesia species was present in the 'western piroplasm' group and was related to Babesia conradae. Phylogenetic analysis of the cox1 sequences indicated possible eastern and western genetic variants for the three Babesia sensu stricto species. Additional analyses are needed to characterize these novel species; however, this study indicates there are now at least four species of piroplasms infecting raccoons in the United States and Canada (Babesia microti-like sp., Babesia lotori, a novel Babesia sensu stricto sp., a novel western Babesia sp.) and a possible fifth species (Babesia sensu stricto) in raccoons in Japan.

A novel mosquito ubiquitin targets viral envelope protein for degradation and reduces virion production during dengue virus infection.

BACKGROUND: Dengue virus (DENV) is a mosquito-borne flavivirus that causes significant human disease and mortality in the tropics and subtropics. By examining the effects of virus infection on gene expression, and interactions between virus and vector, new targets for prevention of infection and novel treatments may be identified in mosquitoes. We previously performed a microarray analysis of the Aedes aegypti transcriptome during infection with DENV and found that mosquito ubiquitin protein Ub3881 (AAEL003881) was specifically and highly down-regulated. Ubiquitin proteins have multiple functions in insects, including marking proteins for proteasomal degradation, regulating apoptosis and mediating innate immune signaling. METHODS: We used qRT-PCR to quantify gene expression and infection, and RNAi to reduce Ub3881 expression. Mosquitoes were infected with DENV through blood feeding. We transfected DENV protein expression constructs to examine the effect of Ub3881 on protein degradation. We used site-directed mutagenesis and transfection to determine what amino acids are involved in Ub3881-mediated protein degradation. Immunofluorescence, Co-immunoprecipitation and Western blotting were used to examine protein interactions and co-localization. RESULTS: The overexpression of Ub3881, but not related ubiquitin proteins, decreased DENV infection in mosquito cells and live Ae. aegypti. The Ub3881 protein was demonstrated to be involved in DENV envelope protein degradation and reduce the number of infectious virions released. CONCLUSIONS: We conclude that Ub3881 has several antiviral functions in the mosquito, including specific viral protein degradation. GENERAL SIGNIFICANCE: Our data highlights Ub3881 as a target for future DENV prevention strategies in the mosquito transmission vector.

Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.

Apurinic/apyrimidinic endonuclease 2 is necessary for normal B cell development and recovery of lymphoid progenitors after chemotherapeutic challenge.

B cell development involves rapid cellular proliferation, gene rearrangements, selection, and differentiation, and it provides a powerful model to study DNA repair processes in vivo. Analysis of the contribution of the base excision repair pathway in lymphocyte development has been lacking primarily owing to the essential nature of this repair pathway. However, mice deficient for the base excision repair enzyme, apurinic/apyrimidinic endonuclease 2 (APE2) protein develop relatively normally, but they display defects in lymphopoiesis. In this study, we present an extensive analysis of bone marrow hematopoiesis in mice nullizygous for APE2 and find an inhibition of the pro-B to pre-B cell transition. We find that APE2 is not required for V(D)J recombination and that the turnover rate of APE2-deficient progenitor B cells is nearly normal. However, the production rate of pro- and pre-B cells is reduced due to a p53-dependent DNA damage response. FACS-purified progenitors from APE2-deficient mice differentiate normally in response to IL-7 in in vitro stromal cell cocultures, but pro-B cells show defective expansion. Interestingly, APE2-deficient mice show a delay in recovery of B lymphocyte progenitors following bone marrow depletion by 5-fluorouracil, with the pro-B and pre-B cell pools still markedly decreased 2 wk after a single treatment. Our data demonstrate that APE2 has an important role in providing protection from DNA damage during lymphoid development, which is independent from its ubiquitous and essential homolog APE1.

Atypical GLUT1 deficiency with prominent movement disorder responsive to ketogenic diet.

Glucose transport protein deficiency due to mutation in the GLUT1 gene is characterized by infantile onset and chronic seizure disorder, microcephaly, global developmental delays, and hypoglycorrhachia. We describe a 10-year-old normocephalic male with prominent ataxia, dystonia, choreoathetosis, and GLUT1 deficiency whose motor abnormalities improved with a ketogenic diet. We illustrate the motor abnormalities, at baseline and after ketogenic diet, that characterize this unusual case. This case broadens the phenotype of GLUT1 deficiency and illustrates the importance of cerebrospinal fluid (CSF) evaluation in detecting potentially treatable conditions in children with undiagnosed movement disorders.

Molecular determinants of sugar transport regulation by ATP.

Intracellular ATP inhibits human erythrocyte net sugar transport by binding cooperatively to the glucose transport protein (GluT1). ATP binding produces altered transporter affinity for substrate and promotes substrate occlusion within a post-translocation vestibule formed by GluT1 cytosolic domains. The accompanying paper (Cloherty, E. K., Levine, K. B., Graybill, C., and Carruthers, A. (2002) Biochemistry 41, 12639-12651) demonstrates that reduced intracellular pH promotes high-affinity ATP binding to GluT1 but inhibits ATP-modulation of GluT1-mediated sugar transport. The present study explores the role of GluT1 residues 326-343 (a proposed GluT1 ATP-binding site subdomain) in GluT1 ATP binding by using alanine scanning mutagenesis. Cos-7 and HEK cells were transfected with a cDNA encoding full-length human GluT1 terminating in a carboxyl-terminal hemagglutinin (HA)-His6 epitope. The transporter (GluT1.HA.H6) is expressed at the surface of both cell-types and is catalytically active. In HEK cells, both parental GluT1- and GluT1.HA.H6-mediated sugar transport are acutely sensitive to cellular metabolic inhibition. Isolated, detergent-solubilized GluT1.HA.H6 is photolabeled by [gamma-32P]-azidoATP in an ATP-protectable manner. Alanine substitution of E329 or G332/R333/R334 enhances GluT1.HA.H6 [gamma-32P]azidoATP photoincorporation but blocks acute modulation of net sugar transport by cellular metabolic inhibition. These actions resemble those of reduced pH on ATP binding to and modulation of red cell GluT1. It is proposed that cooperative nucleotide binding to GluT1 and nucleotide modulation of GluT1-mediated sugar transport are regulated by a proton-sensitive saltbridge (Glu329-Arg333/334).

Cooperative nucleotide binding to the human erythrocyte sugar transporter.

The human erythrocyte glucose transport protein (GluT1) is an adenine nucleotide binding protein. When complexed with cytosolic ATP, GluT1 exhibits increased affinity for the sugar export site ligand cytochalasin B, prolonged substrate occlusion, reduced net sugar import capacity, and diminished reactivity with carboxyl terminal peptide-directed antibodies. The present study examines the kinetics of nucleotide interaction with GluT1. When incorporated into resealed human red blood cell ghosts, (2,3)-trinitrophenyl-adenosine-triphosphate (TNP-ATP) mimics the ability of cytosolic ATP to promote high-affinity 3-O-methylglucose uptake. TNP-ATP fluorescence increases upon interaction with purified human red cell GluT1. TNP-ATP binding to GluT1 is rapid (t(1/2) approximately 0.5 s at 50 microM TNP-ATP), cooperative, and pH-sensitive and is stimulated by ATP and by the exit site ligand cytochalasin B. Dithiothreitol inhibits TNP-ATP binding to GluT1. GluT1 preirradiation with saturating, unlabeled azidoATP enhances subsequent GluT1 photoincorporation of [gamma-32P]azidoATP. Reduced pH enhances azidoATP photoincorporation into isolated red cell GluT1 but inhibits ATP modulation of sugar transport in resealed red cell ghosts and in GluT1 proteoliposomes. We propose that cooperative nucleotide binding to reductant-sensitive, oligomeric GluT1 is modulated by a proton-sensitive saltbridge. The effects of ATP on GluT1-mediated sugar transport may be determined by the number of ATP molecules complexed with the transporter.