Larval spirurids in a supralittoral amphipod in the north-east of Russia and the identification of the intermediate host of Antechiniella septentrionalis (Spirurida: Acuariidae), parasitic in a tundra vole.

The supralittoral amphipod Traskorchestia ditmari (Derzhavin, 1923) was identified as the intermediate host for Antechiniella septentrionalis Ivanova, Dokuchaev & Spiridonov, 2019, a parasite of the tundra vole Microtus oeconomus and Skrjabinocerca sp. (both Spirurida: Acuariidae) in Magadan Oblast in north-eastern Russia. Joint infection by both larval spirurids was not observed. The infective stage of A. septentrionalis was the encysted larvae, while larvae of Skrjabinocerca sp. were free in the amphipod's coelom. The identity of A. septentrionalis was confirmed using cox1 mtDNA gene analysis, performed on adult stages from a tundra vole and on larvae from amphipods. Possible transmission routes of A. septentrionalis are discussed.

An integrative taxonomic assessment of Procamallanus (Spirocamallanus) huacraensis (Nematoda: Camallanidae), infecting the freshwater catfish Trichomycterus spegazzinii (Siluriformes: Trichomycteridae) in Argentina.

Procamallanus (Spirocamallanus) huacraensis infecting the catfish Trichomycterus spegazzinii from Escoipe River, Salta province (Argentina), is redescribed and genetically characterised for the first time, based on three genetic markers (nuclear 18S and 28S rRNA; cytochrome c oxidase subunit I [cox1] mtDNA). The phylogeny of Camallanidae was also discussed. Morphological evaluation of P. (S.) huacraensis using light and scanning electron microscopy revealed the previously undescribed features: location of deirids, accurate morphology of larvae (L1) and ovijector in females, as well as phasmids in males. Differences were found comparing the newly collected material and the type specimens, probably because the original description lacked detailing. Unfortunately, type specimens of P. (S.) huacraensis were no available for loan. The results of morphological and genetic analyses supported the validity of P. (S.) huacraensis. Inconsistencies regarding the taxonomic identification of species of Camallanidae in GenBank database were noted. Based on the current genetic database of Camallanidae, phylogenetic reconstructions using the 18S rRNA sequences were most consistent, due to the inclusion of higher number of taxa. Procamallanus (S.) huacraensis appeared as sister group of P. (S.) rarus, also isolated from a catfish in a neighbouring region. The order and habitat of hosts were also similar within some well-supported parasite lineages, but without common geographic origin. However, it is still premature to make definitive affirmations regarding the role of such features in the phylogenetic patterns of Camallanidae, given the scarcity of genetic data. The phylogenetic reconstructions also confirmed the artificiality of the morphology-based systematics of the family.

TRANSMISSION OF PTERYGODERMATITES NYCTICEBI IN A COLONY OF GOELDI'S MONKEYS ( CALLIMICO GOELDII) AND EVALUATION OF TREATMENT AND CONTROL.

Over a 2-yr period, four Goeldi's monkeys ( Callimico goeldii) died in a private zoo due to infections with the spirurid nematode Pterygodermatites nycticebi. Therapeutic measures with different anthelmintics were not successful. Due to the severe consequences caused by these infections, different actions were initiated, including sanitation measures and controlling of potential intermediate hosts (coprophagous arthropods). To identify possible intermediate hosts, arthropod species detected in the enclosure-parasite-free German cockroaches ( Blattella germanica), European earwigs ( Forficula auricularia), and rough woodlice ( Porcellio scaber)-were experimentally fed with feces of monkeys with patent P. nycticebi infections, resulting in established infections with third-stage larvae (L3) in roaches and earwigs. Furthermore, spiruroid L3 were detectable in 43% of the roaches and 30% of earwigs caught at the zoo. Polymerase chain reaction and sequence analysis of eggs, larval, and adult stages resulted in identical results, confirming the establishment of the parasite's life cycle in the zoo. This is the first documentation of the vector capacity of the European earwigs for P. nycticebi. As a measure of sanitation, a large part of the enclosure was emptied and cleaned. The Goeldi's monkeys were quarantined and treated with levamisole (7.5 mg/kg sc twice in intervals of 2 wk). Repeated coprologic examinations by zinc chloride flotation were undertaken. After the levamisole therapy, eggs were not found in the feces for 3 mo. However, shortly after resettling the monkeys into the sanitized enclosure, reshedding of small amounts of spirurid eggs was observed, whereupon deworming with levamisole was prescribed several times per year. The sanitation measures and the elimination of the intermediate hosts in a natural enclosure are presented as an example of the long-term controlling of the parasites.

Filarial nematode (order: Spirurida) surveillance in urban habitats, in the city of Pécs (Hungary).

As part of the seasonal mosquito control activities in the city of Pécs (Baranya County, Hungary), a total of 1123 adult female mosquitoes belonging to 18 species (including the invasive species Aedes koreicus) were collected from human-inhabited areas, using CO2-baited traps, during two consecutive years. To survey the presence and prevalence of filarial parasites in these mosquitoes, we performed a molecular survey for filarial DNA, attempted by PCR using generic primers (COI), and followed by DNA sequencing. Filaroid nematode DNA was detected in 4% of investigated mosquito pools. Out of 410 pools, 9 pools of mosquitoes were positive for Dirofilaria repens (Aedes vexans, Aedes koreicus, Coquillettidia richiardii), and/or Dirofilaria immitis (Ae. vexans, Cq. richiardii), and further 8 pools were positive for Setaria tundra (Ae. vexans, Cq. richiardii). Our study provides novel insight for prevalence of filaroid nematodes in mosquitoes occurring in close proximity to humans, thereby highlights the possible human and veterinary health importance of these mosquito species, including the recently introduced invasive mosquito Ae. koreicus.

Habitat use pattern of the giant parasitic nematode Crassicauda magna within the pygmy sperm whale Kogia breviceps.

The giant (>3 m) parasitic nematode Crassicauda magna infects kogiid whales, although only 3 studies to date have provided detailed descriptions of these worms, all based upon fragmented specimens. These fragments were found within the neck region of kogiids, an unusual anatomic site for this genus of parasites. C. magna is a species-specific parasite among kogiids, infecting only pygmy sperm whales Kogia breviceps, and with a primarily cervico-thoracic distribution. To date, however, the pattern of habitat use within the host and transmission path of this parasite remain unknown. We used detailed dissections (n = 12), histological examination of host tissues (n = 2), and scanning electron microscopy of excised nematodes (n = 7) to enhance our understanding of this host-parasite relationship. Results revealed that a critical habitat for the parasite is an exocrine gland in the whale's ventral cervical region. C. magna male and female tails were found intertwined within the glandular lumen, and eggs were observed within its presumed secretion, illuminating the transmission path out of the host. The cephalic ends of these worms were often meters away (curvilinearly), embedded deeply within epaxial muscle. A single worm's complete, tortuous 312 cm course, from the gland to its termination in the contralateral epaxial muscle, is described for the first time. This study also provides the first scanning electron micrographs of C. magna, which illustrate taxonomically important features of the heads and tails of both male and female worms.

A new species of Procyrnea (Nematoda: Habronematidae) parasitic in Rhea pennata (Aves: Rheidae) from Patagonia, Argentina, with a key to species of the genus.

Procyrnea choique n. sp. is described from the lesser rhea, Rhea pennata d´Orbigny (Aves: Rheidae), from the Protected Natural Area Península Valdés, Chubut province, Argentina. The new species resembles P. ficheuri, P. murrayi, P. excisiformis, P. dollfusi, P. haliasturi, P. anterovulvata, P. graculae, P. brevicaudata, P. uncinipenis, P. javaensis, P. ameerae, P. ornata, P. aegotheles, P. spiralis, P. ruschii and P. aptera, mainly in the absence of lateral alae; but differs from its congeners by having a left spicule without barbs, the position of the vulva which is post-equatorial, the absence of lateral ridges, absent median precloacal papilla, asymmetrical caudal alae, males more than 6 mm long, left spicule 1 mm long and spicule ratio 1:3. Our results extend the taxonomy of Procyrnea Chabaud, 1958 and comprise the first report of a habronematid from R. pennata. A key to species of Procyrnea is presented.

The Behavior Response of Amphipods Infected by Hedruris suttonae (Nematoda) and Pseudocorynosoma sp. (Acanthocephala).

The manipulation of intermediate host behavior may increase chances of parasite transmission to the definitive host. In freshwater environments of the Neotropical Region, studies on behavioral manipulations by parasites are rare, and the majority of these consider only a single parasite species and/or 1 life stage of a particular parasite species. In Andean Patagonian lakes of Argentina, the amphipod Hyalella patagonica is infected by larvae of the fish nematode Hedruris suttonae and by the bird acanthocephalan Pseudocorynosoma sp. The 3 objectives of the present study were to determine whether H. suttonae and Pseudocorynosoma sp. differ in their effects on behavior of H. patagonica , whether such modification is associated with parasite development, and to assess the associations between behavioral traits. From naturally parasitized amphipods, activity (swimming levels) and phototaxis (light preference) was measured. Only in phototaxis trials did larvae of H. suttonae induce significantly higher levels of photophilia, suggesting that they are manipulative. Scores of activity and phototaxis were positive and significantly related for non-parasitized female amphipods and for amphipods parasitized by larvae of Pseudocorynosoma sp. but were not associated in amphipods parasitized with larvae of H. suttonae (infective and non-infective), suggesting that infection separated the relationship between these variables.

Detailed morphological description of Habronema clarki Foster & Chitwood, 1937, a nematode parasite of capybaras Hydrochoerus hydrochaeris (Linnaeus, 1766) in Brazil.

The genus Habronema has four valid species, of which only two are properly known. The present study aimed to describe in detail the morphology of Habronema clarki through optical and scanning electron microscopy analyses. Our results showed that the labial morphology of this parasite is closer to H. muscae than to H. microstoma. Even so, the characteristic pseudolabia and the slightly convex border of the dorsal and ventral lips are sufficient to distinguish these nematodes. Additional morphological data are presented, thus contributing to the knowledge on this little known nematode. In addition, this study provides new locality records for this species.

Macroscopic lesions of the ventriculus of Rhea americana , Linnaeus, 1758 (Aves: Rheidae) naturally infected by Sicarius uncinipenis (Molin, 1860) (Nematoda: Habronematidae).

There are few studies concerning the parasites of rheas. However, parasitism is the major cause of the limited success in captive breeding of these birds. Deletrocephalus dimidiatus, Deletrocephalus cesarpintoi, Paradeletrocephalus minor, and Sicarius uncinipenis are the most prevalent nematode species affecting these birds, but the lesions caused by these parasites have not been previously reported. Four adult rheas were necropsied to determine the presence or absence of gross lesions within the gastrointestinal tract, associated with parasitic infection. Two rheas parasitized by S. uncinipenis had ulcers on the koilin layer or had parasites penetrating this layer, resulting in widespread necrosis and hemorrhagic areas, whereas the 2 nonparasitized birds did not present lesions. The degree of injury was proportional to the parasitic load found in the birds. Thus, high parasitic loads can result in necrosis of the ventriculus, which may contribute to the death of birds, resulting in economic losses in the rural production of these birds.

Detailed morphological description of Habronema clarki Foster & Chitwood, 1937, a nematode parasite of capybaras Hydrochoerus hydrochaeris (Linnaeus, 1766) in Brazil / Descrição morfológica detalhada de Habronema clarki Foster & Chitwood, 1937, um nematódeo parasita de capivaras Hydrochoerus hydrochaeris (Linnaeus, 1766) no Brasil

The genus Habronema has four valid species, of which only two are properly known. The present study aimed to describe in detail the morphology of Habronema clarki through optical and scanning electron microscopy analyses. Our results showed that the labial morphology of this parasite is closer to H. muscae than to H. microstoma. Even so, the characteristic pseudolabia and the slightly convex border of the dorsal and ventral lips are sufficient to distinguish these nematodes. Additional morphological data are presented, thus contributing to the knowledge on this little known nematode. In addition, this study provides new locality records for this species.

O gênero Habronema tem quatro espécies válidas, das quais apenas duas são propriamente conhecidas. O presente estudo visa descrever em detalhes a morfologia de Habronema clarki por meio de microscopia eletrônica de varredura e de luz. Os resultados demonstram que a morfologia labial do parasita é mais próxima de H. muscae que de H. microstoma. Ainda assim, os pseudolábios característicos e a borda discretamente convexa dos lábios dorsal e ventral são suficientes para se diferenciar esses nematódeos. Dados morfológicos adicionais são apresentados, contribuindo ao conhecimento deste nematódeo pouco conhecido. Em adição, este estudo representa um novo registro de localidade para a espécie.

The effect of a nematode parasite on feeding and dung-burying behavior of an ecosystem engineer.

Dung beetles (genus Phanaeus) consume feces in both their larval and adults forms and because of their unique dietary niche, and behaviors associated with the burial of feces, are considered ecosystem engineers. In addition, because these insects subsist on a diet composed exclusively of feces, it is likely they encounter parasitic propagules more frequently than other animals do. Parasites often alter their host's behavior, so we set out to test whether Physocephalus sexalatus (a cosmopolitan nematode parasite of ungulates) does so in ways that affect the dung beetle's role as an ecosystem engineer and/or its predator-prey relationships (transmission of the parasite). Classic tests of anti-predator behavior did not reveal behavioral differences based on the beetles' infection status. However, this parasite did alter the beetles' behaviors in ways that could be critical for its role in fecal processing and therefore ecosystem engineering. Infected beetles exhibited anorexic behavior and consumed only half the amount of feces ingested by similar uninfected beetles. Infected beetles also buried less feces and did so in tunnels that were significantly shorter than those created by uninfected beetles. Fecal burial is naturally beneficial because it aerates the soil, incorporates nitrogenous compounds, and increases the flow of water thereby making soil and pastureland more productive. We showed that the nematode parasite P. sexalatus itself becomes an ecosystem engineer as it modifies the behavior of its already influential intermediate host.

Incidence of Cercopithifilaria bainae in dogs and probability of co-infection with other tick-borne pathogens.

BACKGROUND: Cercopithifilaria bainae is a filarioid parasite that infects dogs, being transmitted by Rhipicephalus sanguineus group ticks in many countries of the Mediterranean basin. This study assessed the incidence density rate (IDR) of infection by C. bainae in dogs and the probability of co-infection with other tick-borne pathogens (i.e., Anaplasma platys, Babesia vogeli and Hepatozoon canis), in an area of high endemicity in southern Italy. METHODOLOGY/PRINCIPAL FINDINGS: From March 2011 to October 2012, a field study involving 58 young dogs naturally exposed to tick infestation was conducted. Skin and blood samples obtained from each dog six times during an 18-month period were tested for C. bainae by parasite detection within skin snip sediments, with subsequent confirmation through PCR and DNA sequencing. Dogs examined monthly for ticks and A. platys, B. vogeli and H. canis were microscopically and/or molecularly diagnosed and after the first and the second summer seasons, the IDR for positive animal-month at risk was 3.8% and 1.7% in November 2011 and October 2012, respectively. All 58 C. bainae-infected dogs were simultaneously infected with at least one other tick-borne pathogen. After the first summer season (assessment in November 2011), a C. bainae-infected dog had a 33% probability of being infected with H. canis or A. platys, whereas after the second tick season (assessment in October 2012) the probability of co-infection was 78%, 22% and 11% for H. canis, A. platys and B. vogeli, respectively. CONCLUSIONS: Our data indicate that tick-infested dogs are at risk of acquiring infection by C. bainae. In addition, the detection of C. bainae microfilariae indicates a prior tick exposure and, should stimulate testing for other tick-borne disease causing pathogens.

Tick vectors of Cercopithifilaria bainae in dogs: Rhipicephalus sanguineus sensu lato versus Ixodes ricinus.

Recently, dermal microfilariae of a Cercopithifilaria species (Spirurida, Onchocercidae), namely Cercopithifilaria bainae , were detected in dogs from several geographical areas of the Mediterranean basin. Evidence from both laboratory and field studies support the role of the brown dog tick, Rhipicephalus sanguineus sensu lato, as an intermediate host of this nematode. In the present study, we investigated the competence of Ixodes ricinus nymphs as vectors of C. bainae. On November 2012, fully engorged nymphs of I. ricinus (n = 174) and R. sanguineus s.l. (n = 10) were collected from a dog infected by C. bainae. The presence of C. bainae in I. ricinus was assessed by both microscopic dissection of specimens and detection of nematode DNA (PCR), at days 3, 10, 20 and 30 (T1-T4) post-collection; due to the small number of specimens available, R. sanguineus s.l. were examined using the same methods at T4 only. No developing larva of C. bainae was detected in I. ricinus specimens at different time points (T1-T4), even if four of these specimens were PCR-positive at T1. Seven out of ten R. sanguineus s.l. were positive for C. bainae third-stage larvae (L3) at both microscopical and molecular analysis at T4. This study indicates that C. bainae does not develop in I. ricinus nymphs, which preclude the role of this tick as an intermediate host of this parasite. Data presented herein provide new insights into the biology of this filarioid species and will lead to a better understanding of the role of different tick species as vectors of nematodes.

Arthropod intermediate hosts of Abbreviata antarctica (Nematoda: Physalopteridae) in Australia.

This study examines potential arthropod intermediate hosts for the nematode Abbreviata antarctica. Five species of arthropod (tropical native cricket Teleogryllus oceanicus; native roach Drymoplaneta communis; native termite Nasutitermes exitiosus; and 2 introduced species, the East African roach Naupheta cinerea and the Asian cricket Acheta domesticus) were fed feces from a skink, Tiliqua rugosa, containing embryonated eggs of A. antarctica . The insects were dissected at 4 intervals (1 day, 4 days, 8-16 days, and 25-26 days). Viable third-stage larvae were recovered from cysts on the external wall of the midgut and hindgut of 62.5% nymphal and adult T. oceanicus crickets at 25-26 days post-infection and from a single nymphal A. domesticus cricket. No roaches contained eggs or first-stage larvae after 48 hr, and neither eggs nor larvae were found in termites.

Development of some larval nematodes in experimental and natural animal hosts: an insight into development of pathological lesions vis-a-vis host-parasite interactions.

Infective third-stage larvae of three spiruroid nematodes, Ascarops strongylina and Physocephalus sexalatus of pigs and Spirocerca lupi of dogs, were recovered from 14 species of coprophagous beetles belonging to 4 different genera. These larvae were fed to rabbits and/or guinea pigs to study their development in these experimental hosts. Larvae of A. strongylina reached the adult stage in all rabbits and one guinea pig. The adult worms recovered in these hosts were 40% and 4%, respectively, and became diminutive in comparison to their natural hosts. The larvae of P. sexalatus became reencysted in the gastric wall of rabbits inducing marked pathological changes. The infective larvae of S. lupi became reencapsulated in the stomach wall of the rabbit and also showed development in the aortic wall. Adults of Toxocara canis of dog, collected from 5 different regions of the Indian subcontinent, varied significantly in size. The mouse passage of infective larvae of one of these types led to the recovery of the adults from the experimental dogs that were smaller in size and caused severe pathology in natural experimental hosts. Developmental effects shown in experimental hosts and host specificity are of value in understanding the evolution of nematode parasitism.

RNAi mediated silencing of ATPase RNA helicase gene in adult filarial parasite Brugia malayi impairs in vitro microfilaria release and adult parasite viability.

The DExD/H box families of RNA helicases are a multifunctional group of proteins involved in unwinding of inter- and intra-molecular base-paired regions. Successful knockdown of DEAD box RNA helicase gene (BmL3-Helicase) of human lymphatic filarial parasite Brugia malayi was done with specifically designed and chemically synthesized siRNA of <20bp to observe the role of enzyme in parasite biology and its worth as an antifilarial drug target. We made efforts to deliver siRNA into parasite by both electroporation and soaking that resulted into diminished helicase gene expression associated with decreased parasite motility, viability (97%) and release of microfilariae (81.0% reduction) from adult females in vitro. The specific gene knockdown also resulted into death of adult male worms in addition to phenotypic deformities in female worm intrauterine stages. RT-PCR of siRNA treated worms revealed a complete knockdown of BmL3-Helicase transcription within 16h. The present findings thus illustrate that targeting helicase gene of B. malayi would not only interfere with embryogenesis and microfilarial production but also result into decreased motility and viability of microfilariae and adult parasites. The B. malayi helicase enzyme thus represents a possible antifilarial drug target.

Variation in the sex ratio of Rhabdochona fortunatowi (Spirurida: Rhabdochonidae) in Capoeta capoeta gracilis (Cypriniformes: Cyprinidae), relative to levels of infection, host size and temperature.

In the present study, 81 specimens of Capoeta capoeta gracilis (Keyserling, 1861) were collected from the Shiroud River, the Caspian drainage basin, Iran in June and October 2007. The fish were examined for infection with the nematode Rhabdochona fortunatowi (Dinnik, 1933). Nematodes were counted and sexed, then the sex ratio and intestinal site preference of the R. fortunatowi individuals and the intersexual interactions of the parasites were investigated. Significant differences were observed in the prevalence and intensity of infection in relation to host size and sampling season, but not host sex. Parasite burden (mean intensity) was higher in October than in June and the results revealed a decrease in female-to-male sex ratio (FMR) in the fish in October compared to the ones in June. In other words, as the mean intensity of infection of R. fortunatowi increases, the proportion of male worms increases, too. The female-biased sex ratio in June was replaced by a male-biased one in October. Nematode distribution rose to a significant peak in the mid-region of the fish intestine relative to the anterior and posterior parts. Some changes in the distribution of male and female worms in different parts of the host intestine were also observed during June and October. Density-dependent selection and intersexual competition seem to be the main factors driving such a shift in the sex ratio and its variation in different parts of the host intestine.

Host size- and habitat-dependent intensity of Heliconema longissimum (Nematoda: Physalopteridae) in the Japanese eel (Anguilla japonica).

Heliconema longissimum (Ortlepp, 1923) is an ecologically poorly known nematode found in the stomach of Japanese eels, Anguilla japonica Temminck and Schlegel. The occurrence of this nematode in Japanese eels was surveyed in 2 contiguous brackish-water areas (Misho Cove and the lower Renjoji River) of Ehime Prefecture, western Japan, during April 2008 to March 2009. The factors associated with the nematode intensity were also assessed by applying generalized additive models (GAM). Heliconema longissimum exhibited nearly 100% prevalence in both areas, but its intensity differed. The heavier infection in the cove eels indicates that H. longissimum is mainly distributed in the cove, which supports the past speculation for this nematode as a brackish-water parasite. The intensity also increased with the body size of eels. This tendency suggests that the eels inhabiting the cove consume, as they grow, a greater quantity of crustaceans that presumably serve as the nematode's intermediate hosts.

Histopathological manifestations in kidney of Clarias batrachus induced by experimental Procamallanus infection.

Kidney of Clarias batrachus infected with Procamallanus showed varying degrees of histopathological alterations on 15, 30, 45 and 60 days post-infection. The infected kidney showed variable sized glomeruli, cloudy swelling in tubules, vacuolar/atrophic degeneration, fibrosis, mild degenerative changes in distal convoluted tubules, enlarged Bowmen's capsule, necrotic changes as well as increased granulation and hyperplasia in proximal convoluted tubules after 15 days. After 30 days of infection, the changes were rupture of Bowmen's capsule wall, degenerative changes, edema, necrosis, pyknosis, karyorrhexis and karyolysis in proximal and distal convoluted tubules, fibrosis, cloudy swelling and inflammatory lymphocytes, proliferation and shrinkage in glomeruli, and vacuolization in proximal convoluted tubules as well as cloudy swelling. After 45 days, the infected kidney showed cloudy swelling in glomeruli as well as variation in their size, infiltration of RBCs in intralobular vein and necrosis in proximal convoluted tubules, cloudy swelling in interstitium, vacuolization in the epithelial lining cells, necrosis in haemopoietic tissue and inflammatory edema. After 60 days post-infection, the changes were rupture of intralobular vein, cloudy swelling, necrosis in few proximal convoluted tubules, atrophy and shrinkage in glomeruli, distinct inflammatory edema, pyknosis, karyorrhexis and karyolysis, aggregation of lymphocytes and dilation in blood vessels.

Mosquito-based survey for the detection of flaviviruses and filarial nematodes in Aedes albopictus and other anthropophilic mosquitoes collected in northern Italy.

In order to optimize resources, an assay allowing the detection of several etiological agents would be useful. This paper describes a polymerase chain reaction (PCR)-based assay for the parallel detection of different pathogens in mosquitoes (Diptera: Culicidae). The assay combines the analysis of both RNA and DNA, hence allowing the detection of both flaviviruses (Flaviviridae: Flavivirus) and filarial nematodes (Spirurida: Onchocercidae). The sensitivity of diagnostic assay for both flavivirus and nematode infections was tested by means of spiked samples and resulted adequate for the purpose of screening mosquito samples. The assay was implemented to check pathogen infections in 637 mosquitoes field-collected in Romagna, northern Italy. The integrity of RNA extracted from mosquito pools was checked by control amplifications targeting the 18 S rRNA of mosquitoes. Control amplifications were successful in 118 out of 119 pools. Flavivirus RNA was not found in any of these 118 pools, whereas DNA of Dirofilaria immitis (Leidy) was detected in one pool of Aedes albopictus (Skuse).