Sviluppo di mezzi e sistemi di sorveglianza e monitoraggio dei ditteri culicidi di importanza sanitaria

Nel corso degli ultimi anni le problematiche legate al ruolo vettore delle zanzare stanno emergendo sia per quanto riguarda l’uomo che gli animali allevati e selvatici. Diversi arbovirus come West Nile, Chikungunya, Usutu e Dengue, possono facilmente spostarsi a livello planetario ed essere introdotti anche nei nostri territori dove possono dare avvio a episodi epidemici. Le tecniche di monitoraggio e sorveglianza dei Culicidi possono essere convenientemente utilizzate per il rilevamento precoce dell’attività virale sul territorio e per la stima del rischio di epidemie al fine dell’adozione delle opportune azioni di Sanità Pubblica. Io scopo della ricerca del dottorato è inserito nel contesto dei temi di sviluppo del Piano regionale sorveglianza delle malattie trasmesse da vettori in Emilia Romagna. La ricerca condotta è inquadrata prevalentemente sotto l’aspetto entomologico applicativo di utilizzo di dispositivi (trappole) che possano catturare efficacemente possibili insetti vettori. In particolare questa ricerca è stata mirata allo studio comparativo in campo di diversi tipi di trappole per la cattura di adulti di zanzara, cercando di interpretare i dati per capire un potenziale valore di efficacia/efficienza nel rilevamento della circolazione virale e come supporto alla pianificazione della rete di sorveglianza dal punto di vista operativo mediante dispositivi adeguati alle finalità d’indagine. Si è cercato di trovare un dispositivo idoneo, approfondendone gli aspetti operativi/funzionali, ai fini di cattura del vettore principale del West Nile Virus, cioè la zanzara comune, da affiancare all’unica tipologia di trappola usata in precedenza. Le prove saranno svolte sia in campo che presso il laboratorio di Entomologia Medica Veterinaria del Centro Agricoltura Ambiente “G. Nicoli” di Crevalcore, in collaborazione con il Dipartimento di Scienze e Tecnologie Agroambientali della Facoltà di Agraria dell’Università di Bologna.

Population structure of the peridomestic mosquito Ochlerotatus notoscriptus in Australia

Ochlerotatus notoscriptus (Skuse) (Diptera: Culicidae) is the predominant peridomestic mosquito in Australia where it is the primary vector of dog heartworm, Dirofilaria immitis (Leidy), and a potentially important vector of arboviruses (Barmah Forest, Ross River) with geographical variation of vector competence. Although widespread, Oc. notoscriptus has low dispersal ability, so it may have isolated subpopulations. The identification of gene flow barriers may assist in understanding arbovirus epidemiology and disease risk, and for developing control strategies for this species. We investigated the population structure of Oc. notoscriptus from 17 sites around Australia, using up to 31 putative allozyme loci, 11 of which were polymorphic. We investigated the effect of larval environment and adult morphology on genetic variation. At least five subpopulations were found, four in New South Wales (NSW) and one unique to Darwin. Perth samples appear to be a product of recent colonization from the Australian east coast. For NSW sites, a Mantel test revealed an isolation by distance effect and spatial autocorrelation analysis revealed an area of effective gene flow of 67 km, which is high given the limited dispersal ability of this species. No consistent difference was observed between 'urban' and 'sylvan' habitats, which suggests frequent movement between these sites. However, a finer-scaled habitat study at Darwin revealed small but significant allele frequency differences, including for Gpi. No fixed allozyme differences were detected for sex, size, integument colour or the colour of species-diagnostic pale scales on the scutum. The domestic habit of Oc. notoscriptus and assisted dispersal have helped to homogenize this species geographically but population structure is still detectable on several levels associated with geographical variation of vector competence.

Flavivirus isolations from mosquitoes collected from Saibai Island in the Torres Strait, Australia, during an incursion of Japanese encephalitis virus

Adult mosquitoes (Diptera: Culicidae) were collected in January and February 2000 from Saibai Island in the Torres Strait of northern Australia, and processed for arbovirus isolation during a period of Japanese encephalitis (JE) virus activity on nearby Badu Island. A total of 84 2 10 mosquitoes were processed for virus isolation, yielding six flavivirus isolates. Viruses obtained were single isolates of JE and Kokobera (KOK) and four of Kunjin (KUN). All virus isolates were from members of the Culex sitiens Weidemann subgroup, which comprised 53.1 % of mosquitoes processed. Nucleotide sequencing and phylogenetic analysis of the pre-membrane region of the genome of JE isolate TS5313 indicated that it was closely related to other isolates from a sentinel pig and a pool of Cx. gelidus Theobald from Badu Island during the same period. Also molecular analyses of part of the envelope gene of KUN virus isolates showed that they were closely related to other KUN virus strains from Cape York Peninsula. The results indicate that flaviviruses are dynamic in the area, and suggest patterns of movement south from New Guinea and north from the Australian mainland.

Development time and survival of Verrallina funerea (Theobald) (Diptera : Culicidae) immatures and other brackish water mosquito species in southeast Queensland, Australia

Verrallina funerea (Theobald) is a brackish water mosquito that is recognised as an important pest and vector in southeast Queensland, Australia. Immature development time and survival of Ve. funerea was defined in the laboratory in response to a range of temperatures (17-34 degrees C) and salinities (0-35 parts per thousand (p.p.t)). The expression of autogeny in this species was also assessed. Salinity only had a slight effect on mean development time from hatching to adult emergence (7.0-7.4 d at salinities of 0, 17.5 and 31.5 p.p.t) and survival was uniformly high (97.5-99.0%). Mean development times were shorter at 26, 29 and 32 degrees C (7.0, 6.8 and 6.8 d, respectively) and longest at 17 degrees C (12.2 d). The threshold temperature (t) was 5.8 degrees C and the thermal constant (K) was 142.9 degree-days above t. Survival to adulthood decreased from > 95% (at 17-29 degrees C) to 78% (at 32 degrees C) and 0% (at 34 degrees C). No expression of autogeny was observed. Immature development times of Ve. funerea, Ochlerotatus vigilax (Skuse) and Oc. procax (Skuse) were then determined under field conditions at Maroochy Shire. Following tide and rain inundation, cohorts of newly hatched larvae were monitored daily by dipping, and time until pupation was noted. Tidal inundation triggered hatching of Ve. funerea and Oc. vigilax larvae whereas Oc. procax larvae were found only after rain inundation. Estimates of Ve. funerea and Oc. vigilax field development times were similar (8-9 d) while Oc. procax development time was slightly longer (9-10 d). Based on these survey results, control activities targeting Ve. funerea must be initiated 4 d (if using Bacillus thuringiensis var. israelensis de Barjac) or 5 d (if using s-methoprene) after inundation. However, Casuarina glauca Sieber canopy and branchlets covering breeding habitats may present a problem for the penetration of such treatments.

Risks for ross river virus disease in tropical Australia

Background There are no analytical studies of individual risks for Ross River virus (RRV) disease. Therefore, we set out to determine individual risk and protective factors for RRV disease in a high incidence area and to assess the utility of the case-control design applied for this purpose to an arbovirus disease. Methods We used a prospective matched case-control study of new community cases of RRV disease in the local government areas of Cairns, Mareeba, Douglas, and Atherton, in tropical Queensland, from January I to May 31, 1998. Results Protective measures against mosquitoes reduced the risk for disease. Mosquito coils, repellents, and citronella candles each decreased risk by at least 2-fold, with a dose-response for the number of protective measures used. Light-coloured clothing decreased risk 3-fold. Camping increased the risk 8-fold. Conclusions These risks were substantial and statistically significant, and provide a basis for educational programs on individual protection against RRV disease in Australia. Our study demonstrates the utility of the case-control method for investigating arbovirus risks. Such a risk analysis has not been done before for RRV infection, and is infrequently reported for other arbovirus infections.

West Nile and St. Louis encephalitis virus antibody seroconversion, prevalence, and persistence in naturally infected pig-tailed macaques (Macaca nemestrina)

Pig-tailed macaques (Macaca nemestrina) naturally infected with West Nile virus were monitored from 1999 to 2005 to determine virus-specific antibody seroconversion, prevalence, and persistence. Antibodies persisted for up to 36 months, as detected by epitope-blocking enzyme-linked immunosorbent and hemagglutination inhibition assays. Exposure to cocirculating St. Louis encephalitis virus was evaluated by Western blotting and immunofluorescence assays.

Diagnóstico laboratorial de casos de importação de infeções por vírus Zika

O vírus Zika (Flavivirus) é um arbovírus transmitido sobretudo por mosquitos, mas também, por transmissão materno-fetal e sexual. Existem evidências que as infeções por vírus Zika podem estar associadas à síndrome de Guillian-Barré e a casos congénitos de microcefalia e outras malformações do sistema nervoso central. As infeções por vírus Zika, Dengue e Chikungunya partilham, atualmente, os mosquitos vetores, a sintomatologia e a distribuição geográfica. O Centro de Estudos de Vetores e Doenças Infeciosas do Instituto Nacional de Saúde Doutor Ricardo Jorge no seu Laboratório Nacional de Referência de Vírus Transmitidos por Vetores tem desenvolvido o diagnóstico e estudos epidemiológicos de vírus transmitidos por artrópodes desde o princípio dos anos 90. O diagnóstico de Zika foi desenvolvido e padronizado em 2007. O laboratório desenvolveu testes de diagnóstico molecular e serológico tendo identificado vários casos de importação para o território português e feito o diagnóstico diferencial com Dengue e Chikungunya e o despiste de infeção em grávidas e em casos de transmissão sexual.

Identification of Hotspots in the European Union for the Introduction of Four Zoonotic Arboviroses by Live Animal Trade

Live animal trade is considered a major mode of introduction of viruses from enzootic foci into disease-free areas. Due to societal and behavioural changes, some wild animal species may nowadays be considered as pet species. The species diversity of animals involved in international trade is thus increasing. This could benefit pathogens that have a broad host range such as arboviruses. The objective of this study was to analyze the risk posed by live animal imports for the introduction, in the European Union (EU), of four arboviruses that affect human and horses: Eastern and Western equine encephalomyelitis, Venezuelan equine encephalitis and Japanese encephalitis. Importation data for a five-years period (2005-2009, extracted from the EU TRACES database), environmental data (used as a proxy for the presence of vectors) and horses and human population density data (impacting the occurrence of clinical cases) were combined to derive spatially explicit risk indicators for virus introduction and for the potential consequences of such introductions. Results showed the existence of hotspots where the introduction risk was the highest in Belgium, in the Netherlands and in the north of Italy. This risk was higher for Eastern equine encephalomyelitis (EEE) than for the three other diseases. It was mainly attributed to exotic pet species such as rodents, reptiles or cage birds, imported in small-sized containments from a wide variety of geographic origins. The increasing species and origin diversity of these animals may have in the future a strong impact on the risk of introduction of arboviruses in the EU.

Evidence of cryptic genetic lineages within Aedes notoscriptus (Skuse)

Aedes notoscriptus (Skuse), a mosquito from the southwest Pacific region including Australia, has been implicated as a vector of arboviruses, but its status as a species is unclear. To investigate the taxonomic situation, we assessed genetic variation and phylogenetic relationships among Ae. notoscriptus from the east coast of Australia, Western Australia and New Zealand. Phylogenetic analyses of DNA sequence data from mitochondrial markers indicate that Ae. notoscriptus is a complex of divergent genetic lineages, some of which appear geographically restricted, while others are widespread in eastern Australia. Samples from New Zealand and Western Australia were related to populations from one southern Australian lineage. Nuclear markers show no evidence of genetic isolation by geographic distance in the overall sample of mosquitoes, but strong isolation by distance is obvious within two of the lineages, supporting their status as isolated gene pools. The morphological character of wing centroid size variation is also associated with genetic lineage. These findings point to the possibility that Ae. notoscriptus is a complex of species, highlighting the need to understand physiological and ecological differences that may influence future control strategies.

Follicular dendritic cell disruption as a novel mechanism of virus-induced immunosuppression

Arboviruses (Arthropod-borne viruses) cause acute diseases that are increasingly affecting both human and animal health. Currently, there is a critical lack of understanding about the nature of arbovirus-host interactions in the lymph nodes (LNs), the place where the adaptive immune response is initiated and shaped. In this study, we used bluetongue virus (BTV) and its natural sheep host, to characterise the early events of an arbovirus infection with particular focus on the LNs. Our findings reveal a previously uncharacterized mechanism used by an arbovirus to manipulate host immunity. This study shows that BTV, similarly to other antigens delivered through the skin, is transported rapidly via the lymph to the peripheral lymph nodes. Here, BTV infects and disrupts the stromal network of marginal reticular cells and follicular dendritic cells composing the scaffolding of the follicular area. These cells contribute to antigen presentation and affinity maturation of B-cells for the production of antibodies. Consequently, we observed a loss of germinal centre structure, which hinders B-cell proliferation. This process results in a delayed production of high affinity and virus neutralizing antibodies that is directly related to the virulence of the BTV strain used and the severity of disease. Moreover the humoral immune response to a different antigen is also hampered in BTV-infected animals. Our data show that an arbovirus can evade the host antiviral responses by inducing an acute immunosuppression. Although transient, this immunosuppression occurs at the critical early stages of infection when a delayed host humoral immune response likely affects virus systemic dissemination and the clinical outcome of disease.