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.     

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.     

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.     

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.     

Potential vectors of Dirofilaria immitis in Macon County, Alabama

A study was conducted in Macon County, Alabama (east-central) during the mosquito breeding seasons of 1978 and 1979 to determine the potential vector(s) of Dirofilaria immitis. Live mosquitoes were collected with CO2-baited, miniature light traps, and small hand aspirators. These mosquitoes were subsequently dissected to determine whether immature stages of the heartworm were present. Specific communities were selected based on their having at least 4 known cases of D immitis infection present in the local dog population. All positive collections were made between the months of May and September. Of 326 Anopheles punctipennis females dissected, 34 were positive, and of 84 Aedes vexans 6 were positive for infective (L3) larvae. Of 520 Culex quinquefasciatus females tested, 14 contained 2nd-stage larvae only. Immature stages of D immitis were collected during most of the mosquito breeding season. Data collected during the survey indicate that An punctipennis and to a lesser extent, Ae vexans are probably the principal vectors of D immitis in Macon County, Ala.     

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.     

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.     

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.     

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.     

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.     

A duplex real-time polymerase chain reaction assay for the detection of and differentiation between Dirofilaria immitis and Dirofilaria repens in dogs and mosquitoes

This study describes a duplex real-time polymerase chain reaction (PCR) assay for the detection and differentiation between Dirofilaria immitis and Dirofilaria repens in dog blood and mosquitoes. Regions of a cytochrome oxidase 1 (cox1) mitochondrial DNA fragment and the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA were amplified from microfilariae and adult worm samples, using a sensitive SsoFast? EvaGreen(?) based real-time PCR method coupled with melting-curve analysis. The limit of the real-time PCR in detecting microfilaria and adult worm DNA was also tested both in dog blood and in artificially infected microfilarial. Two peaks at different melting temperatures (T(m)) for D. immitis (mean ± SD=75.7 ± 0.3°C) and D. repens (mean ± SD=70 ± 0.7°C), respectively, were obtained for microfilarial and adult positive controls of both species when examined separately and together. The real-time PCR protocol was also efficient in detecting microfilarial and adult DNA of both species when tested in samples spiked with DNA from Aedes albopictus, in Aedes aegypti experimentally infected by D. repens and in Culex pipiens naturally infected by D. repens and D. immitis. The high sensitivity of real-time PCR confirmed its reliability in detecting small amounts of genomic DNA either in dog blood or mosquitoes (2.5 pg/μl and 3 × 10(-1)pg/μl for D. immitis and D. repens, respectively). This assay is proposed as a tool for the epidemiological surveillance of the two most important Dirofilaria species in areas where they are endemic and sympatric.     

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.     

Detection of Theileria and Babesia species in ticks collected from cattle

The present study was carried out to detect tick species that infest cattle, and Theileria and Babesia species transmitted by these ticks in Kayseri province (Turkey). A total of 300 cattle were examined for tick infestations. Of the 300 cattle, 117 (39%) were infested with ticks. A total of 1160 ticks belonging to 11 Ixodid genera were collected from the infested animals and their shelters. The most prevalent tick species was Boophilus annulatus 26.37% (306/1160) followed by Hyalomma marginatum marginatum 21.12% (245/1160) and Rhipicephalus turanicus 18.7% (217/1160). The collected ticks were separated into 43 tick pools, according to their species. These pools were examined for bovine Theileria and Babesia species (Theileria sp., Babesia sp., Theileria annulata, T. buffeli/orientalis, Babesia bigemina, B. bovis and B. divergens) by using the reverse line blotting method (RLB). Of the 43 tick pools examined, 6 (14%) were infected with B. bigemina, 4 (9.3%) with T. annulata, and 1 (2.3%) with Babesia sp., whereas 1 (2.3%) displayed mixed infection with T. annulata + B. bigemina. The sequence and phylogenetic analyses of Babesia sp., which could not be identified to the species level by RLB, were performed. In the phylogenetic tree, Babesia sp. (Kayseri 1) grouped with Babesia sp. (Kashi 2), Babesia sp. (Kashi 1), Babesia sp. (Xinjiang) and B. orientalis with 96.8-100% identity.     

Seroepidemiologic survey of Dirofilaria immitis infection among domestic dogs in Taipei City and mountain aboriginal districts in Taiwan (1998-1999).

To estimate the seroprevalence of Dirofilaria immitis infection in domestic dogs in Taiwan, we utilized a commercial ELISA kit (Snap, IDEXX, USA) for detecting circulating antigens released by adult female worms. Serum specimens of 664 domestic dogs sampled from Taipei City in northern Taiwan and 14 mountain aboriginal districts in eastern Taiwan were screened for D. immitis antigens. Multivariate-adjusted odds ratios (ORs) with their 95% confidence intervals (CIs) were estimated by multiple logistic regression analysis. A total of 89 subjects were antigen-positive, giving a seroprevalence of 13.4%, of which the seroprevalence in Taipei City and mountain aboriginal districts were 13.8 and 12.1%, respectively. The mean overall seropositive rates were 6.3% in 1-3-year-old age group, 14.1% in 3-6-year-old age group and 23.7% in the > or =6-year-old age group. The older the age, the higher the seroprevalence (OR=4.6, 95% CI=2.4-9.0 for the > or =6-year-old age group versus 1-3-year-old age group, P<0.001) for all the dogs in the present study. Moreover, seroprevalence was not different between female and male dogs in either Taipei City or mountain aboriginal districts (P>0.05). Also, no significant difference in seroprevalence among dogs between the two geographical areas was found (P>0.05). In the logistic regression analysis, the seroprevalence of D. immitis remained significantly increased with age after multivariate adjustment in the present study. To our knowledge, this is the first report on the status of D. immitis infection in domestic dogs in Taipei City and mountain aboriginal districts in Taiwan to date.     

A survey of ixodid ticks feeding on cattle and prevalence of tick-borne pathogens in the Black Sea region of Turkey

The study reports the frequency of infestation and the prevalence of tick-borne pathogens in feeding adult ticks detached from cattle in two climatic zones of the Black Sea region of Turkey. A total of 2160 adult ticks were collected during 2007-2008. Of these, 1062 were randomly selected, divided into 224 pools, and tested for the presence of bovine Theileria, Babesia, and Anaplasma species. Eleven tick species were recognized on cattle in the study. Hyalomma marginatum was widely disrubuted in the semi-arid bioclimatic zone, but few specimens were collected in the humid bioclimatic zone. The most prevalent tick species in the humid climatic zone was Ixodes ricinus. Infection rates were calculated as the maximum likelihood estimation with 95% confidence intervals (CI). Overall, 4% (CI 2.87-5.44) of 224 tick pools were found to be positive for the pathoges by Reverse line blot. Maximum likelihood estimation of the infection rate varied among tick species, ranging from 2.68% (CI 0.16-12.68) in Haemaphysalis sulcata to 10.49% (CI 4.07-23.66) in Rhipicephalus bursa. The most prevalent tick-borne pathogen was Anaplasma phagocytophilum at 6.78% (CI 3.41-12.18) followed by A. centrale (6.56%, CI 0.42-31.47), Anaplasma/Ehrlichia spp. (3.61%, CI 1.99-6.06), Babesia spp. (3.33%, CI 1.65-6.03), and T. buffeli/orientalis (2.71%, CI 0.73-7.18). Sequencing results indicated that Babesia spp. shared 99% to 100% similarity with the unnamed Babesia sp. Kashi 1 and 2, Babesia sp. Kayseri 1 and Babesia sp.CS58. Anaplasma/Ehrlichia spp. were 98% and 100% identical to Ehrlichia canis and Ehrlichia sp. Omatjenne strain, respectively.     

Distribution, prevalence, and relative risk of filariasis in dogs from the State of Washington (1997-1999).

Using antigen capture and filter tests, 6,078 dogs throughout the state of Washington were examined for filariasis between July 1, 1997 and October 31, 1999. In western Washington, 791 males and 901 females examined were outdoors, not on prophylaxis, and had traveled out of the state; 6/791 (0.8%) males and 7/901 (0.8%) females were infected with Dirofilaria immitis (D. immitis), and one (0.1%) male and one (0.1%) female were infected with Dipetalonema reconditum (D. reconditum). There were also 392 males and 362 females examined that were outdoors, not on prophylaxis, and had not traveled out of western Washington. One (0.1%) female was infected with D. immitis, and two (0.5%) males and one (0.2%) female were infected with D. reconditum. In eastern Washington, 707 males and 826 females examined were outdoors, not on prophylaxis, and had traveled out of the state; 9/707 (1.0%) males and 4/826 (0.5%) females were infected with D. immitis, and no D. reconditum was found. There were also 376 males and 412 females examined that were outdoors, not on prophylaxis, and had not traveled out of the state. Three (0.8%) males and three (0.7%) females had D. immitis. One (0.2%) female had D. reconditum. Distribution of D. immitis-infected, nontravel dogs in eastern Washington was only found between 120 degrees and 119 degrees west longitude in the communities of Richland, Moses Lake, Okanogan, and Omak. Enzootic transmission of D. immitis and D. reconditum is occurring in both eastern and western Washington.     

Canine dirofilariosis in two cities of southeastern Spain

Several cases of human subcutaneous/ocular dirofilariosis caused by Dirofilaria repens from an area in the southeastern Spain where previous epidemiological studies have shown a very low prevalence of this species in dogs, have been studied in our laboratory. Since the prevalence of this species in dogs did not correspond to the incidence of human cases in the zone studied, a preliminary epidemiological survey was carried out on 114 dog blood samples from two kennels and one veterinary clinic. Knott, polymerase chain reaction (PCR) with specific primers for Dirofilaria immitis and D. repens, and ELISA with adult E/S D. immitis and adult somatic D. repens antigens for the detection of specific IgG, were used. Fifty-three out of the 114 samples analyzed were positive for Knott and/or PCR to D. immitis or to D. repens. D. repens was the species with the highest prevalence, 84.6 and 37.1%, respectively, in each kennel. IgG antibodies against D. immitis and D. repens, were detected in 11 samples which gave negative results to both Knott and PCR. These results demonstrate a very high prevalence of D. repens that could be associated with the increasing incidence of human subcutaneous/ocular cases recently detected in this zone.     

Prevalence of Dirofilaria immitis infection in dogs from Dandong, China

The aim of the present study was to estimate the prevalence and risk factors of Dirofilaria immitis infection in dogs from Dandong, China. A total of 886 dogs were examined for D. immitis infection by microscopic examination and PCR, indicating that the prevalence was 16.6% (213/886) and 24.0% (147/886), respectively. The odds of infection were significantly higher in older dogs and dogs sheltered in outdoor, compared to the younger ones and ones sheltered in indoor. No significant difference of infection was observed in different genders, and between pure breed and cross-breed dogs in the same rearing conditions. These results indicated that the risk of exposure to D. immitis in dogs is high in Dandong, China, and prophylaxis against the parasite is advisable to decrease the incidence of canine dirofilariosis.