Aedes vexans and Culex pipiens as the potential vectors of Dirofilaria immitis in Central Turkey

This study was carried out to investigate the potential vectors and relative mosquito infection rates of Dirofilaria immitis throughout two mosquito seasons (2008-2009) in Kayseri province where is located in Central Anatolian part of Turkey. For this aim, totally 1198 genomic DNA pools, extracted and grouped according to the species and collection site (1-17 specimens/pool) from 6153 mosquito specimens, were examined by PCR using species-specific primers for D. immitis. The captured mosquitoes from 46 focuses were survived under in vitro conditions for 7 days to allow the development of larval stages of D. immitis. DNA extraction was performed individually to both thorax-head and abdomens in order to determine infective and infected mosquito specimens, respectively. The most abundant mosquito species in the study area was determined as Ae. vexans (51.7%) and this was followed by Cx. pipiens (42.1%), Cx. theileri (3.1%), Cs. annulata (1.5%), An. maculipennis (1.0%) and Cx. hortensis (0.6%). The PCR results indicated that 9/312 and 12/312 pools from Ae. vexans abdomens and thorax-heads were positive for filarial DNAs, respectively where as 3/241 pools of each abdomens and thorax-heads from Cx. pipiens were positive for D. immitis DNAs. The minimum infection rates (MIRs) for Ae. vexans and Cx. pipiens were calculated as 0.41 and 0.12, respectively. Although D. immitis DNA's were found in both pools from Ae. vexans and Cx. pipiens, the calculated MIRs provide evidence that Ae. vexans could be the main potential vector of D. immitis in Kayseri.     

Molecular survey of Dirofilaria immitis and Dirofilaria repens by direct PCR for wild caught mosquitoes in the Republic of Korea

Adult mosquito collections using New Jersey light traps and Black-hole light traps were conducted to determine the potential vectors and the relative mosquito infection rates of Dirofilaria immitis and Dirofilaria repens in Gyeonggi and Gangwon Provinces, Republic of Korea, 2005. Dirofilaria spp. were confirmed by polymerase chain reaction (PCR) using species-specific primers for D. immitis and D. repens. Minimum field infection rates (MFIR) [MFIR = (number infected pools/total number of mosquitoes) x 1000] of 12 pools/2059 total number mosquitoes (5.8) and 21 pools (10.1) of the wild caught mosquitoes were positive by PCR for D. immitis and D. repens, respectively. Dual infections of both D. immitis and D. repens were detected by PCR in eight pools (3.9). Anopheles sinensis sensu lato (includes An. sinensis s.s., An. pullus, An. kleini, An. belenrae, and An. lesteri), An. sineroides, Aedes vexans nipponi, Culex pipiens and Armigeres subalbatus were found to be infected and are potential vectors of D. immitis and D. repens in Korea. Infections observed in mosquitoes represent potential health risks for domestic animal and human populations.     

Dirofilariasis in Argentina: historical review and first report of Dirofilaria immitis in a natural mosquito population

Argentina is one of the four South American countries where the presence of Dirofilaria immitis is currently confirmed. The objective of this study was to review information on dirofilariasis in the country, and to report our recent findings on mosquito vectors. Since the first report of dogs with unidentified microfilariae in 1926, D. immitis was found in seven provinces and canine prevalence ranged 0-71% at local scale. National prevalence was 8% by the end of the 1980s and current information is available only for Buenos Aires Province. Four pulmonary human infections of D. immitis and one subcutaneous of Dirofilaria sp. were documented. The common coati was the only wild host found, and natural infection in mosquitoes was not previously reported in the country. In our recent mosquito survey in Greater Buenos Aires, we captured and dissected 2380 mosquitoes belonging to 20 species. According to a minimum temperature of 14 degrees C, the potential transmission period (PTP) for D. immitis in Buenos Aires covers 6 months, and the most favourable period (mean temperature above 20 degrees C) takes place from the middle of November to the beginning of April. To identify potential vectors of the parasite, we assessed weekly abundances of mosquito species during those PTP estimated previously. We found two specimens of Culex pipiens and one of Aedes aegypti carrying non-infective stages of D. immitis. These two highly anthropophilic mosquitoes may enhance the role of D. immitis as zoonotic agent in temperate Argentina.     

Canine dirofilariasis in the canton of Ticino and in the neighboring areas of northern Italy

The distribution of canine dirofilariosis in Southern Ticino (Switzerland) and in the neighbouring provinces of Varese and Como (Italy) was investigated. Blood samples were collected from 308 dogs which had remained in the local area and were outdoor-housed, older than 1.5 years and had not been treated previously with preventive or microfilaricidal drugs. Microfilariae of Dirofilaria immitis and D. repens were found in 33 (10.7%) and 17 (5.5%) dogs, respectively. Ten more dogs (3.2%) tested positive for circulating antigens. Four infected dogs lived in Southern Ticino: two harboured D. immitis, one D. repens and one had a mixed infection. In addition, 3887 mosquitoes were captured in five sample sites by means of dog-baited traps. Culex pipiens, Aedes geniculatus and Ae. vexans were the most abundant species. Infective stages of D. immitis were observed in local strains of Ae. geniculatus and Cx. pipiens, following engorgement on a microfilaraemic dog and the successive rearing in laboratory conditions. A chemo-prophylactic scheme in four administrations between July and October is recommended for Southern Switzerland.     

Dirofilaria immitis and its potential mosquito vectors in central New York State.

Screening of 25,822 dog blood samples indicated approximately a 1% infection rate with Dirofilaria immitis. Dipetalonema reconditum microfilariae were found in approximately 2% of 1,876 feral dogs examined. Laboratory experimentation indicated that 6 of 10 local mosquito species examined allowed successful extrinsic incubation of D immitis. Indices of experimental infection indicated Aedes triseriatus and Anopheles quadrimaculatus were excellent hosts for D immitis, but other factors considered, Aedes vexans probably served as the primary vector of D immitis. Coquillettidia perturbans and Culex pipiens-restuans group were considered unsuitable hosts and thus unlikely vectors of canine heartworm. Natural filarial infections were found in An quadrimaculatus and Ae vexans.     

Mosquito vectors of dog heartworm in the United States: vector status and factors influencing transmission efficiency

Dog heartworm (Dirofilaria immitis) is dependent on mosquito vectors for its maintenance and transmission among vertebrate hosts. Consequently, D. immitis abundance and distribution are closely linked with mosquito vector biology and ecology. Information on the important dog heartworm vectors in the United States is limited and no comprehensive surveillance of dog heartworm in US mosquitoes has been undertaken to date. Here, we review information gleaned from a number of field surveys documenting heartworm presence in wild mosquito populations as well as laboratory assessments of mosquito vector capacity. Various biological and ecological factors likely contribute to the relative importance of different vector species. We describe some of these factors, rank the leading criteria for efficient vectors, and present the most likely vector species found across the United States. Considering the recent emergence of drug resistance among D. immitis strains, practical knowledge of heartworm vector biology and control should be incorporated into heartworm disease management programs. We conclude by proposing that heartworm control would benefit by targeting mosquito vectors, and we suggest ways in which veterinarians can incorporate the recognition of vector importance into heartworm prevention recommendations imparted to clients.     

Mosquito collection in endemic areas of Japanese encephalitis in Hokkaido, Japan

A study was conducted during 1985 and 1986 to evaluate the roles of mosquito species as possible vectors of Japanese encephalitis (JE) virus in Hokkaido. The number of Culex tritaeniorhynchus was very low among the four pig farms where outbreaks of abortion caused by JE virus were observed in swine populations. At one farm near Sapporo, only one Cx. tritaeniorhynchus was found among a total of 510 mosquitoes collected during the survey period from July to October 1985, even when JE virus activity among sentinel pigs was revealed by seroconversion. At another farm in the south, no individuals of this mosquito species were found among 987 mosquitoes collected at the time of the outbreaks of abortion. Cx. pipiens pallens, Anopheles species, Aedes vexans nipponii, and Ae. japonicus were predominant over Cx. tritaeniorhynchus. Cx. tritaeniorhynchus, almost a solve vector species of JE virus in the southern part of Japan, is probably not a vector of the virus in Hokkaido. The collected mosquitoes (2,332 from 1985 and 1,403 from 1986) were processed for virus isolation but no JE virus was isolated. More extensive field studies are necessary to provide further information on the role of mosquito species other than Cx. tritaeniorhynchus in the transmission of JE virus in the northern limits of its range including Hokkaido.     

Competence of Aedes albopictus and Culex quinquefasciatus as vector of Dirofilaria immitis after blood meal with different microfilarial density

Aedes albopictus and Culex quinquefasciatus were fed canine blood with different microfilarial density of Dirofilaria immitis ranging from 2500 to 25,000 mff/ml. Larval development in these two mosquito species did not differ significantly. Although C. quinquefasciatus ingested more microfilariae, the number of larvae which developed in A. albopictus was invariably greater than in C. quinquefasciatus. Mortality of the engorged A. albopictus was significantly greater than that of C. quinquefasciatus, and higher microfilarial density raised the mortality in both species. The vector efficiency index of A. albopictus was greater than C. quinquefasciatus at all microfilarial densities, but its survival time was much reduced. Thus, dogs with low microfilarial density are implicated as the main source for the transmission of D. immitis from dogs to mosquitoes.     

Changing climate and changing vector-borne disease distribution: the example of Dirofilaria in Europe

Climatic changes, together with an increase in the movement of dogs across Europe, have caused an increase in the geographical range of Dirofilaria infections. The present paper is focuses on northeastern European countries, where survey data have shown an increase of Dirofilaria repens infections both in animals and humans. A growing degree day-based forecast model has been developed to predict the occurrence. The model is based on evidence that there is a threshold of 14 °C below which Dirofilaria development will not proceed in mosquitoes, there is a requirement of 130 growing degree-days (GDDs) for larvae to reach infectivity, and there is a maximum life expectancy of 30 days for a mosquito vector. The output of this model predicted that the summer temperatures (with peaks in August) are sufficient to facilitate extrinsic incubation of Dirofilaria even at latitudes of 56 °N and longitudes of 39 °E. Despite the fact that both Dirofilaria immitis and D. repens have the same temperature requirement for extrinsic incubation in mosquitoes, empirical data has shown that D. repens is the main cause of dirofilarial infections in both humans and animals. Clinical signs are absent in most canine infections with D. repens. Furthermore, diagnosis is problematic and in-clinic serological tests, such as those for D. immitis, do not exist. Therefore, most infections go undiagnosed, allowing the infection to spread undetected.     

Prevalence and epidemiological aspects of Dirofilaria immitis in dogs from Kayseri Province, Turkey

This study was conducted to determine the prevalence of Dirofilaria immitis infection and to investigate the risk factors related to heartworm disease in dogs from Kayseri, Turkey. Blood samples were collected from 280 dogs from May 2005 to March 2006 and were examined by membrane filtration-acid phosphatase histochemical staining and antigen Elisa techniques to detect circulating microfilariae and antigens of D. immitis, respectively. Of the total of 280 dogs, 27 were positive for D. immitis with a prevalence value of 9.6%. In addition 29.6% of positive dogs determined to have occult D. immitis infections. D. immitis was the only canine filarial parasite present in the study area. The mean number of microfilariae in infected dogs was 4730+/-5479 per ml of blood. The highest heartworm prevalence were observed in 7-10 age group (28.6%) followed by 4-6 (17.1%) and 0.5-3 (4.8%) age groups. The differences between 0.5-3 and other age groups were found significant, whereas no statistically significant difference was observed between 4-6 and 7-10 age groups. The infection was more prevalent in males, larger breeds and the dogs not on prophylaxis. No statistically significant difference was observed between stray and owned dogs. Our results suggest that heartworm treatment and prophylaxis should be considered in Kayseri Province.     

Aedes albopictus is a natural vector of Dirofilaria immitis in Italy

Investigations were carried out in Padova town (Veneto region, NE Italy) to define the actual role of Aedes albopictus in the natural transmission of Dirofilaria nematodes, and to assess the risk that its presence might represent for veterinary and medical health. During summer 2000-2002 daytime captures of human-attracted mosquitoes were carried out in three areas of the town. The presence of filarial parasites in mosquitoes was evaluated by PCR, and sequencing confirmed species assessment. DNA extraction was performed separately on pools of the insect abdomen and thorax-head, to discriminate between Dirofilaria infected/infective specimens. A total of 2721 mosquitoes were caught and A. albopictus was the most abundant species (2534). Filarial DNA was found in 27.5% (19/69) of the abdomen pools formed with mosquitoes collected in summer 2000, and in 11.1% (16/144) and 4.9% (6/123) thorax-head pools coming from samplings 2001 and 2002, respectively. Filarial DNA was belonging to D. immitis and all studied areas harboured infective specimens. These results prove A. albopictus as natural vector of D. immitis in Italy. Moreover, they support the hypothesis that the presence of the mosquito could affect the transmission pattern of canine heartworm disease in urban environment and, considering the aggressive anthropophylic behaviour of the species (30-48 bites/h) proven in Padova town, could enhance the circulation of filarial nematodes from animals to humans.     

Simulium jenningsi Malloch (Diptera: Simuliidae): a vector of Onchocerca lienalis Stiles (Nematoda: Filarioidea) in New York

Biting flies were collected from the umbilical areas of Onchocerca lienalis-infected cattle in New York (state) from June through September of 1980. Of the 766 flies collected, 705 were Simulium jenningsi. Microfilariae were detected in the midguts of 37 (50%) of 73 females dissected immediately after the flies had fed. The mean number of larvae per positive fly (fly with microfilariae) was 15.2. Third-stage larvae were recovered from 25 (21.9%) of 114 S jenningsi dissected 8 to 13 days after they had fed on the infected cattle; the mean number of 3rd-stage larvae per positive fly was 3.5. Dissections of flies performed on days 1 through 7 after feeding yielded various numbers of 1st and 2nd-stage larvae from the thoracic muscles. Ovarian dissections performed on 304 S jenningsi attacking cattle indicated an overall parous rate of 58%. Naturally occurring infections with filarial larvae indistinguishable from O lienalis were found in 7.3% of the parous females. Three of these flies, or 1.7% of the parous collection, harbored 3rd-stage larvae. The onset of naturally occurring filarial infections in the population of S jenningsi coincided with a peak in the parous rate in late June. Thereafter, filarial infections were generally detected when the parous rate was above 50%.     

Limited efficacy of levamisole against adults of Dirofilaria immitis in a dog

A dog with chronic dirofilariasis (Dirofilaria immitis) was given 15 doses of 12 or 24 mg of levamisole/kg of body weight in 6 treatments within 96 days. Concentration of microfilariae in the blood was determined before and after treatment, and Aedes triseriatus mosquitoes were fed on the dog 1 to 5 days after each treatment and examined for D immitis larvae. Several adult worms were recovered from the dog 160 days after the end of the treatments.     

Detection and molecular characterization of avian Plasmodium from mosquitoes in central Turkey

Assessing vector-parasite relationship is important in understanding the emergence of vector-borne diseases and the evolution of parasite diversity. This study investigates avian Plasmodium parasites in mosquitoes collected from Kayseri province in Central Anatolian, Turkey and determines the haemosporidian parasite lineages from these mosquito species. A total of 6153 female mosquitos from 6 species were collected from 46 sites during June-August of 2008 and 2009. Each mosquito's head-thorax and abdomen were separated, categorized with respect to species and collection area and pooled for DNA extraction. A total of 1198 genomic DNA pools (599 thorax-head, 599 abdomen) were constituted of which 128 pools (59 thorax-head, 69 abdomen) were positive for avian haemosporidian parasites (Plasmodium and Haemoproteus) by Nested-PCR analysis. Culex pipens, Aedes vexans, Culex theileri and Culiseta annulata were positive with minimum infection rates (MIRs) of 16.22 and 18.15, 4.72 and 5.98, 5.18 and 10.36, 10.64 and 10.64 in their thorax-head and abdomen parts, respectively. No avian haemosporidian DNA was detected from Culex hortensis and Anopheles maculipennis. Phylogenetic analyses of the partial cytb gene of avian haemosporidian mt-DNA from 13 positive pools revealed that 11 lineages in four phylogenic groups were Plasmodium and the other two were Haemoproteus. Our results suggest that Cx. pipiens could probably be the major vector of avian Plasmodium in Central Turkey. This is the first report of molecular detection and characterization of avian Plasmodium lineages from mosquitoes in Turkey.     

Prevalence of Dirofilaria immitis infection among shelter cats

OBJECTIVE: To determine prevalence of Dirofilaria immitis infection among shelter cats. DESIGN: Cross-sectional study. ANIMALS: 239 cats euthanatized at an animal shelter in southeastern Michigan. PROCEDURE: A gross necropsy focusing on the thoracic cavity, heart, lungs, and pulmonary vessels was performed within 5 hours after cats were euthanatized. Blood was collected directly from the heart of cats found to be infected and tested, using a filter test for microfilariae. Serum was tested for D immitis antigens by use of 2 antigen detection kits and for D immitis-specific antibodies by use of 2 antibody detection kits. RESULTS: Cats ranged from approximately 4 months to 15 years old. Adult D immitis were found in 6 (2.5%) cats. Blood could not be recovered from 1 of the cats with heartworm infection. For the 5 other cats, results of the filter test were negative, and results of both antigen and both antibody tests were positive. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that cats living in an urban area in the northern part of the United States have a low prevalence of adult D immitis infection. However, this is likely to be an underestimate of the true prevalence of infection, because no attempts were made to identify cats infected with larval or juvenile stages of D immitis.     

Microscopic, serologic and molecular surveys on Dirofilaria immitis in stray dogs, Turkey

The prevalence of Dirofilaria immitis infection was evaluated in stray dogs of Erzurum, Turkey. A total of 123 whole-blood and 93 sera samples were collected from stray dogs older than 6 months were lived in animal shelter. The PCR and direct microscopic examinations were used for the detection of microfilaria and indirect-ELISA was performed for the detection of anti-D. immitis antibodies. The prevalence of D. immitis in the canine population was 8.1% by PCR, 2.1% by ELISA. In addition, microfilaria burdens of Dirofilaria sp. was 4.8% by direct blood smear examination. There was a statistical difference (P=0.05) in the prevalence between males (10.5%) and females (2.3%) by direct blood smear examination. Similarly there was a statistical difference (P<0.05) in the prevalence between males (15.8%) and females (4.7%) by PCR. Dogs belonging to the 0.5-1 years old group showed the highest prevalence than 2-4 ages group with three tests. Among the 93 samples screened by the ELISA, two samples were positive for the D. immitis antibodies. Both positive dogs with ELISA were females.     

Prevalence of canine dirofilariosis in the City of Buenos Aires and its outskirts (Argentina)

During the past 10 years, the prevalence of canine dirofilariosis in the City of Buenos Aires and its outskirts, particularly in the northern and southern areas, has increased significantly. In the present work, studies were carried out in dogs living in the city and in its northern, western and southern outskirts from 1997 to 2001. For this purpose, 782 blood samples were collected and analyzed to determine circulating antigen, processed with the Witness Merial antigen test. The samples resulted in negative tests for subjects who lived in the city; however, 17.7 and 23.5% of the tests were positive from the northern and southern outskirts, respectively. When analyzed by sex, positive results were distributed as follows: 62.5% males and 37.5% females (P<0.05). No significant statistical difference was found on comparing purebred and cross-breds (P<0.05). It is interesting to point out the geographical distribution of the disease, which confirms that ecological factors such as water currents, abundant vegetation and the existence of mosquitoes all year round, are important for the biological cycle of Dirofilaria immitis.     

Immunoblot analysis of specific antigen bands predictable for Dirofilaria immitis infection in cats

Serial sera from four mongrel cats experimentally inoculated with infectious larvae of Dirofilaria immitis were analyzed by immunoblot patterns against a phosphate buffered saline-extract of D. immitis. Antigen-specific protein bands detected indicate that the low molecular weight bands of 36, 32, 22, 19 and 14 kDa, are predictable for positive adult worm infection, suggesting diagnostic usefulness for adult D. immitis infection in cats.     

The incrimination of Aedes (stegomyia) aegypti as the vector of Dirofilaria repens in Nigeria

Six local species of culicides were identified as the common mosquitoes in Zaria, out of 15 species captured using various adult and larval collection methods. These common culicides are Culex pipiens fatigans, Anopheles gambiae grp., Mansonia africana, Culex pipiens pipiens, Aedes (stegomyia) aegypti and Aedes vittatus. They were each fed directly on a local dog naturally infected with Dirofilaria repens to evaluate their refractoriness/susceptibility to dirofilarial infection. In a number of donor-feeding trials, 39. 4% Culex pipiens fatigans; 58.9% An gambiae grp.; 60.5% Mansonia africana; 1.8% of Culex pipiens pipiens; 23.4% Ae aegypti and 3.3% of Ae vittatus successfully fed on the microfilaraemic host. Only Aedes aegypti was susceptible to the infection as all 40 (100%) Ae aegypti reaching 10-14 day post-blood meal had infective (L(3)) larvae of D. repens. The remaining five species were refractory. The microfilariae in the five non-susceptible mosquitoes were always found trapped in the blood meal in the insects midgut (stomach). These trapped microfilaria were dead by the 2nd day in the insect's midgut. However, in the susceptible Ae aegypti, the microfilariae were set free from the blood meal in the midgut and within 24h migrated to the malpighian tubules (MT) of the mosquitoes. All Ae aegypti dissected 5-7 day post-infective blood meal showed the typical quiescent sausage stage (L(2)) larvae in the malpighian tubules. At day-10 post-blood meal, relatively active infective (L(3)) larvae of D. repens were found in the MT; and by day 12-14, highly motile infective larvae had reached the insect's head and proboscis, with infective larvae occasionally oozing out during dissection through the tip of the proboscis. The rate of development of D. repens to infective larvae was faster in mosquitoes infected in July when the environmental temperature was 24.5 degrees C than those infected in November when the temperature was 22.5 degrees C. The latter were delayed for 4 days. The breeding sources of Ae aegypti, the local vector implicated were also identified. As no particular vector of this zoonotic filaria has been identified previously in Nigeria, these findings could make any control programme more focussed and easier.     

Is heartworm disease really spreading in Europe?

Based on recently published surveys and newly acquired data, a study was conducted to verify the distribution of filarial worm (Filarioidea) infections in Europe, with particular emphasis on canine heartworm infection (Dirofilaria immitis). A Geographic Information System based on thermal regimen was constructed as a means to identify areas potentially suitable for heartworm transmission, taking into account that the development of D. immitis larvae in the mosquito does not occur below the threshold temperature of approximately 14 degrees C. Furthermore, a bionomic model of D. immitis in its mosquito vectors, which calculates the moving cumulative heartworm development unit parameter, was applied using the available temperature data to assess the theoretic transmission timing of heartworm in Europe. The results show that the earliest infection risk occurs in Spain on March 21 and the latest risk occurs in Spain on September 11. The longest risk period occurs in Spain (Murcia station: March 21-November 11), and the shortest risk period occurs in northeastern Europe. The study also provides the first risk assessment maps for Europe and suggests that if the actual climatic trend continues, filarial infection should spread into previously infection-free areas.