The enzyme-linked immunosorbent assay for detecting antibodies against Dirofilaria immitis in dogs

Antisera prepared in mice by injection of antigens from Dirofilaria immitis, Toxocara canis, Dipylidium canium and Fasciola hepatica and sera from Dirofilaria-infected and non-infected dogs were tested at different dilutions using an enzyme-linked immunosorbent assay (ELISA). For this ELISA, adult D. immitis antigen was fractionated by gel filtration methods and then absorbed with immunoadsorbent-fixed IgG fractions from mouse sera immunized with various parasites. The results indicated satisfactory discrimination between antisera to D. immitis and those to other parasites. A significant ELISA O.D. value was considered to be greater than or equal to 0.30 with a 1 : 250 dilution of the dog sera. However, the lack of significant differences between the O.D. value of microfilaraemic and amicrofilaraemic infections was observed. These facts suggest that the use of immunoadsorbent chromatography for canine dirofilariasis is especially useful for purifying antigens and eliminating cross-reactions against other parasitic infections, when immunological methods are used for serodiagnosis.     

Canine Dirofilaria immitis infection in a hyperenzootic area: examination by parasitologic findings at necropsy and by two serodiagnostic methods

A total of 174 dogs from an area hyperenzootic for Dirofilaria immitis were grouped into 4 age categories and necropsied; information was obtained on adult D immitis infections and on the presence of microfilariae. Serum samples from these dogs were examined by an enzyme-linked immunosorbent assay (ELISA) for antibody to adult D immitis and by an indirect fluorescent antibody test (IFAT) for antibody to microfilarial surface antigens. In dogs less than or equal to 5 months of age, necropsy demonstrated no evidence of infection; however, positive serologic results indicated that some of these dogs had prepatent infections. The percentage of dogs with ELISA titers (positive) increased with age, as did the percentage of dogs with adult D immitis infections. The IFAT results were positive in some dogs in each age category. Sera from all 29 dogs with occult infections were positive by ELISA. Sera from 6 of 20 dogs with occult dual-sex heartworm infections and 1 of 9 dogs with occult single-sex heartworm infections were positive by IFAT. For diagnosing occult dirofilariasis, the ELISA had a positive predictive value which increased with age of the dog to a maximum of 65.0% in dogs greater than or equal to 12 months of age; ELISA had a negative predictive value of 100% in all age groups. In contrast, positive and negative predictive values for the IFAT decreased with age of the dog to 60% and 37.5%, respectively, in dogs greater than or equal to 12 months of age.     

Serologic prevalence of Dirofilaria immitis,Ehrlichia canis,and Borrelia burgdorferi infections in Brazil

Dogs infected with Dirofilaria immitis, Ehrlichia canis, or Borrelia burgdorferi may show nonspecific clinical signs or may be asymptomatic. In Brazil, E. canis and D. immitis infections are frequently diagnosed based on the presence of classical signs; however, serologic tests are seldom performed to confirm the presence of infection. To estimate the seroprevalence of these three canine diseases in Brazil, 2,553 dogs presented at veterinary practices for various tests, routine treatments, or examinations were evaluated by an in-office commercial ELISA test kit (SNAP 3Dx, IDEXX Laboratories). Each dog was examined by the veterinarian, and a whole-blood sample was collected and immediately tested for the simultaneous detection of B. burgdorferi and E. canis antibodies and D. immitis antigen. D. immitis infection was detected in 51 dogs (2.0%) and E. canis antibodies were present in 505 dogs 19.8%). Only one dog tested positive for B. burgdorferi antibodies.     

The occurrence of Dirofilaria immitis in dogs in South Australia

Heart, lung and samples of blood from 230 dogs were examined for infections of filarial parasites. Dirofilaria immitis worms and microfilariae were detected in one dog. Blood samples from a further 1428 dogs were examined for microfilariae and 22 were found to be infected. Eighteen dogs were infected with D immitis microfilariae and four with Dipetolonema reconditum microfilariae. The histories were available for 18 of the dogs infected with heartworm and only seven dogs had not travelled outside South Australia. It was concluded that heartworm infection was endemic in South Australia but the apparent prevalence was only about 1%.     

What is happening outside North America regarding human dirofilariasis?

The etiologic agents of human dirofilariasis in the Old World are Dirofilaria immitis, which cause pulmonary and subcutaneous nodules, and Dirofilaria repens, which cause ocular lesions. Although reports of new cases of dirofilariasis are sporadic in other parts of the world, a considerable amount of information is generated in Europe regarding human dirofilariasis. Most cases have been detected in the Mediterranean countries, Ukraine, and Russia; however, isolated or short series of cases have been reported in the Balkan Republics and central and northern European countries. Seroepidemiologic studies have provided evidence that humans living in endemic areas present rates of infection similar to those of the autochthonous canine populations. Antibodies against endosymbiont Wolbachia bacteria have been demonstrated recently in human Dirofilaria infections. During D. immitis infections, preadult worms and third- and fourth-stage larvae are often destroyed by the host reaction, releasing a considerable amount of Wolbachia, and a Th1-type response against Wolbachia and/or filarial antigens is mounted. On the contrary, infections with D. repens, in which worms frequently remain intact, no Th1-type response has been observed. As humans are resistant hosts, the Th1-response could have a role in the resistance against parasites. The causes for the rise in the incidence of human dirofilariasis as well as the possible application of Wolbachia antigens in the serodiagnosis of human infections are discussed.     

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.     

Current prevalence of Dirofilaria immitis in dogs, cats and humans from the island of Gran Canaria, Spain

The island of Gran Canaria is a hyperendemic area for canine dirofilariasis. The aim of the present study was to provide data on Dirofilaria immitis in dogs, cats, and humans on this island in 2010. The data confirms the prevalence in the overall canine population (19%), with a considerably higher prevalence (43%) in the autochthonous breed of Canarian Warren hound. The prevalence in the feline population (33%) is higher than that of the canine population, and the existence of specific D. immitis antibodies in the inhabitants of the island of Gran Canaria (12%) is confirmed. In both cats and humans, the prevalence, according to the different climate areas on the island, is related to the prevalence of D. immitis in dogs in the same area, which shows the key epidemiological role played by the canine host with regard to the transmission to other hosts of D. immitis.     

Prevalence of selected zoonotic and vector-borne agents in dogs and cats in Costa Rica

To estimate the prevalence of enteric parasites and selected vector-borne agents of dogs and cats in San Isidro de El General, Costa Rica, fecal and serum samples were collected from animals voluntarily undergoing sterilization. Each fecal sample was examined for parasites by microscopic examination after fecal flotation and for Giardia and Cryptosporidium using an immunofluorescence assay (IFA). Giardia and Cryptosporidium IFA positive samples were genotyped after PCR amplification of specific DNA if possible. The seroprevalence rates for the vector-borne agents (Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis, and Anaplasma phagocytophilum) were estimated based on results from a commercially available ELISA. Enteric parasites were detected in samples from 75% of the dogs; Ancylostoma caninum, Trichuris vulpis, Giardia, and Toxocara canis were detected. Of the cats, 67.5% harbored Giardia spp., Cryptosporidium spp., Ancylostoma tubaeforme, or Toxocara cati. Both Cryptosporidium spp. isolates that could be sequenced were Cryptosporidium parvum (one dog isolate and one cat isolate). Of the Giardia spp. isolates that were successfully sequenced, the 2 cat isolates were assemblage A and the 2 dog isolates were assemblage D. D. immitis antigen and E. canis antibodies were identified in 2.3% and 3.5% of the serum samples, respectively. The prevalence of enteric zoonotic parasites in San Isidro de El General in Costa Rica is high in companion animals and this information should be used to mitigate public health risks.     

Indirect fluorescent antibody test in occult dirofilariasis.

Indirect fluorescent antibody titers to Dirofilaria immitis microfilaria (IFA-mf) and peripheral eosinophilia were recorded from 15 to 52 months in ten experimentally infected dogs with occult dirofilariasis (heartworm infection without microfilaremia). Five dogs which were experimentally sensitized with D immitis microfilaria did not exhibit microfilaremia after inoculation with infective-stage larvae. In three other dogs, microfilaremia suddenly ceased after 4 to 7 months. In these three dogs, antimicrofilarial antibodies were detectable by IFA-mf test as soon as microfilaremia ended. In the remaining two dogs, which exhibited spontaneous occult dirofilariasis, antibodies were detected at the end of the prepatent period of 6 months. The presence of adult worms was confirmed by angiocardiography. Significant IFA=mf titers (greater than or equal to 1:8) persisted after successful treatment with an adulticide. Reinfection of treated dogs reestablished occult dirofilarasis. Eosinophilia was present in all dogs and peaked at about 3, 6, and 9 months after they were inoculated with infective-stage larvae. At necropsy, the ten dogs harbored gravid, reproducing adult worms in the heart and pulmonary arteries.     

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.     

Specificity of scolex and oncosphere antigens for the serological diagnosis of taeniid cestode infections in dogs

Groups of dogs raised free of helminths were monospecifically infected with the common nematodes Toxocara canis, Ancylostoma caninum and Trichuris vulpis. Serums from these dogs, and a group of dogs of unknown history but infected with Dirofilaria immitis and Dipylidium caninum, had levels of antibody to their homologous nematode antigens readily detectable by ELISA. No cross-reactions were apparent when these serums were tested by ELISA using oncosphere antigens of Taenia hydatigena, T. pisiformis and T. ovis, scolex excretory/secretory antigens of T. hydatigena, T. pisiformis and Echinococcus granulosus or protoscolex antigen of E. granulosus.     

Systematic review of the distribution of the major vector-borne parasitic infections in dogs and cats in Europe

On the basis of a systematic review of the literature, this paper provides the first evidence-based maps of the distribution of the major vector-borne parasitic infections of dogs and cats in Europe. From an initial survey of 894 publications, data from 268 were analysed, summarised and collated. Prevalence data were used to calculate the force of infection to provide distribution and incidence maps for canine infections with Leishmania infantum, Dirofilaria immitis, Dirofilaria repens, Babesia canis and Ehrlichia canis. There was little information from some areas but, in spite of its acknowledged incompleteness, the study provides an evidence-based framework upon which to assess the risks of infection, and will provide a basis, by correlation with climatic and vegetation data, to derive more comprehensive risk assessment maps for Europe. To clinicians in both endemic and non-endemic countries, it will be of assistance in decision-making with respect to diagnosis and preventive measures.     

Transmission trials, ITS2-PCR and RAPD-PCR show identity of Toxocara canis isolates from red fox and dog.

Toxocara canis isolates from dog and from red fox were compared in transmission trials and with molecular analysis using RAPD-PCR technique and comparison of the ITS2 sequence. After oral infection of bitches with 20,000 embryonated T. canis eggs of vulpine and canine origin, the vertical transmission to pup's was examined. All animals of both groups developed typical clinical symptoms of toxocarosis. The haematological, serological, parasitological and post mortem results showed no differences between both isolates except for the infectivity of T. canis stages in mice where the fox isolate showed a significant higher infectivity than the dog isolate. The RAPD-PCR showed a similarity coefficient of 0.95, similar to the range of intraspecific variation in Toxocara cati and Toxascaris leonina specimens as outgroups. The ITS2 comparison showed a 100% identity between both isolates with no intraspecific variations. Therefore, the study shows that the fox and the dog isolate of T. canis were identical in infectivity, transmission and molecular structure; a host adaptation could not be found and the fox has to be seen as a reservoir for T. canis infections in dogs. Considering the increasing number of foxes in urban areas the importance of helminth control in dogs is stressed.     

A survey for infection with Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi, and Babesia canis in feral and client-owned dogs in the Turks and Caicos Islands, British West Indies

The frequency of infection with Dirofilaria immitis and Babesia canis and seropositivity to Ehrlichia canis and Borrelia burgdorferi in feral and client-owned dogs was determined. Feral dogs were 14.8 and 11.2 times more likely to be seropositive to D. immitis and E. canis, respectively, than were client-owned dogs. None of the dogs tested positive for B. burgdorferi or B. canis.     

Exposure to infectious agents in dogs in remote coastal British Columbia: Possible sentinels of diseases in wildlife and humans

Ranked among the top threats to conservation worldwide, infectious disease is of particular concern for wild canids because domestic dogs (Canis familiaris) may serve as sources and reservoirs of infection. On British Columbia's largely undeveloped but rapidly changing central and north coasts, little is known about diseases in wolves (Canis lupus) or other wildlife. However, several threats exist for transfer of diseases among unvaccinated dogs and wolves. To gain baseline data on infectious agents in this area, including those with zoonotic potential, we collected blood and stool samples from 107 dogs in 5 remote communities in May and September 2007. Serology revealed that the dogs had been exposed to canine parvovirus, canine distemper virus, Bordetella bronchiseptica, canine respiratory coronavirus, and Leptospira interrogans. No dogs showed evidence of exposure to Ehrlichia canis, Anaplasma phagocytophilum, Borrelia burgdorferi, Dirofilaria immitis, or Cryptococcus gattii. Of 75 stool samples, 31 contained at least 1 parasitic infection, including Taeniid tapeworms, the nematodes Toxocara canis and Toxascaris leonina, and the protozoans Isospora sp., Giardia sp., Cryptosporidium sp., and Sarcocystis sp. This work provides a sound baseline for future monitoring of infectious agents that could affect dogs, sympatric wild canids, other wildlife, and humans.     

Prevalence of dog intestinal parasites and risk perception of zoonotic infection by dog owners in São Paulo State, Brazil

Coprological examination was used to estimate the prevalence of gastrointestinal parasites in stray and domiciled dogs from Botucatu, S?o Paulo State, Brazil. Risk factors for dog infection were assessed in relation to demographic, husbandry and management data. The dog owners completed a questionnaire survey on some aspects of dog parasitism such as parasite species, mechanisms of infection, awareness of zoonotic diseases and history of anthelmintic usage. Parasites were found in the faeces of 138 dogs, with an overall prevalence of 54.3%. Dogs harbouring one parasite were more common (31.4%) than those harbouring two (18.5%), three (3.2%) or four (1.2%). The following parasites and their respective frequencies were detected: Ancylostoma (37.8%), Giardia (16.9%), Toxocara canis (8.7%), Trichuris vulpis (7.1%), Dipylidium caninum (2.4%), Isospora (3.5%), Cryptosporidium (3.1%) and Sarcocystis (2.7%). Stray dogs were found more likely to be poliparasitized (P<0.01) and presented higher prevalence of Ancylostoma, T. canis and Giardia (P<0.01) than domiciled ones. Toxocara canis was detected more frequently in dogs with <6 months of age (P<0.05) and no effect of sex or breed could be observed (P>0.05). Except for Ancylostoma, that showed a significantly higher prevalence in dogs living in a multi-dog household (P<0.01), parasite prevalences were similar in single- and multi-dog household. The answers of dog owners to the questionnaire showed that the majority does not know the species of dog intestinal parasites, the mechanisms of transmission, the risk factors for zoonotic infections, and specific prophylactic measures. The predominance of zoonotic species in dogs in the studied region, associated with the elevated degree of misinformation of the owners, indicates that the risk of zoonotic infection by canine intestinal parasite may be high, even in one of the most developed regions of Brazil.     

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.     

Seroprevalence of Borrelia burgdorferi, Anaplasma phagocytophilum, Ehrlichia canis, and Dirofilaria immitis among dogs in Canada

The seropositivity of dogs to Borrelia burgdorferi, Anaplasma phagocytophilum, and Ehrlichia canis antibodies, and Dirofilaria immitis antigen was assessed in Canada. Borrelia burgdorferi had the highest seroprevalence, while that of Dirofilaria immitis has not changed significantly in the past 20 y. The risk for these vector-borne infectious agents in Canadian dogs is low but widespread with foci of higher prevalence.     

Dirofilaria immitis infection in dogs: cardiopulmonary biomarker levels

Cardiopulmonary biomarkers are biological parameters that can be objectively measured and quantified as indicators of pathogenic processes (heartworm disease) or as indicators of response to therapeutic intervention. To determine levels of cardiopulmonary biomarkers in canine dirofilariasis, measurements of cardiac troponin T, cardiac troponin I, myoglobin, and D-dimer concentrations were performed for dogs with and without evidence of adult heartworm infection. The results showed that levels of cardiac troponin T were undetectable in all dogs studied while levels of cardiac troponin I were higher in dogs infected with Dirofilaria immitis. In healthy dogs, levels of myoglobin and D-dimer were below detection limits of the instrument and were significantly higher in heartworm-infected dogs, notably in microfilaremic dogs. The results suggest the possibility of using troponin I and myoglobin as markers for cardiac damage and the D-dimer as a supportive tool for a diagnosis of pulmonary thromboembolism in dogs with cardiopulmonary dirofilariasis.     

An investigation of the role of immunologic factors in anemia associated with canine heartworm disease

Direct Coombs' antiglobulin tests were performed on 80 dogs with patent Dirofilaria immitis infection and 170 dogs negative for microfilaria of D. immitis. Presence or absence of anemia was determined by hematocrit in 55 of the heartworm negative dogs and 68 of the dogs with heartworm disease. Heartworm infected dogs showed a higher incidence (37%) of anemia than noninfected dogs (14.5%). Anemia was most prevalent in two groups of dogs with heartworm infection, one group showing vena caval syndrome (91%) and the other occult dirofilariasis (62.5%). These latter two groups of dogs also showed a significantly higher number of positive Coombs' reactions at 37 degrees C than other dogs with heartworm disease and the noninfected dogs. The number of positive Coombs' reactions at 4 degrees C among the total of 80 dogs with heartworm infection was significantly higher than that for dogs without heartworm disease. However, there was no positive correlation between anemia and the outcome of the Coombs' test at either temperature. These findings do not suggest that immunologic factors play a major role in the pathogenesis of anemia in dogs with heartworm disease.