A Survey for the Microfilariae of the Canine Heartworm, Dirofilaria immitis, in the Calgary Region of Southern Alberta

A blood survey for the prevalence of the microfilariae of the canine heartworm, Dirofilaria immitis was conducted in the Calgary area of southern Alberta between November 1977 and August 1979. A total of 514 blood samples was examined by the modified Knott's test. All of the samples were negative for D. immitis microfilariae. Wright's stained blood smears taken from 19 animals at the Calgary Zoo also proved negative. One smear from a male two-toed sloth (Choloepus didactylus) contained sheathless microfilariae which were distinguishable from those of D. immitis. These results, as well as mail survey data indicate that D. immitis is not endemic in the Calgary area.

It is recommended that the modified Knott's test be used for similar large scale sampling studies. In addition, it is suggested that the testing of both native and exotic zoo animals which could serve as definitive hosts of D. immitis be continued. These animals may become local sources of infection or introduce other species of microfilariae which will have to be differentiated from those of D. immitis.

     

Heartworm in dogs in Canada in 1988

In late November 1988, 1581 small and mixed animal clinics and institutional veterinarians across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1988, and 46% of them responded. Veterinarians reported that 181,577 dogs were blood-tested for heartworm disease and 367 dogs were found with D. immitis microfilariae. Another 60 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1988 as 441 (0.24%).     

Heartworm in dogs in Canada in 1987

In late November 1987, 1246 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1987, and 50% of them responded. Veterinarians reported that 165,428 dogs were blood tested for heartworm disease and 511 dogs were found with D. immitis microfilariae. Another 78 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1987 as 589 (0.35%).     

First molecular detection of Dirofilaria immitis (Spirurida: Onchocercidae) infection in dogs from Northern Algeria

Dirofilaria immitis and Dirofilaria repens are mosquito-borne filarioid nematodes that affect dogs and other domestic and wild carnivores, causing heartworm disease and subcutaneous dirofilariosis, respectively. In Algeria, the data about the epidemiology of these infections is largely unknown. The present study was designed to establish the occurrence of D. immitis and D. repens in dogs in Algeria using molecular tools.In 2014 and 2015, a total of 209 dogs over one year of age of different breed and sex, living in Northern Algeria, were examined and blood samples were collected from each dog. The presence of D. immitis and D. repens in these samples was detected by real-time PCR followed by standard PCR and sequencing. Overall, the blood of 209 dogs from two departments was collected and only 3 (1.4%) of the blood samples were found positive for D. immitis DNA. Sequencing of the corresponding amplicon displayed a 99.8% identity to D. immitis, confirming the presence of this mosquito-borne nematode in Algeria. Furthermore, all tested samples were negative for D. repens.     

Heartworm in dogs in Canada in 1989

In late November 1989, 1732 clinics and institutional veterinarians were sent a questionnaire to assess the status of Dirofilaria immitis, and 51.7% responded. Of 247,716 dogs tested, 394 had D. immitis microfilariae and 51 were amicrofilaremic for a total of 445 cases and heartworm prevalence of 0.17%. Most (408) of these dogs had no preventive medication and the prevalence among dogs tested and unprotected was 1.01%. That prevalence was considerably higher in endemic areas. Thirty-seven dogs with heartworm had preventive medication. Heartworm was most frequent in companion dogs over three years of age maintained outdoors in rural areas. About 75% of the cases had never left Canada, 26% had clinical signs and 125 were not treated.

Heartworm was reported from British Columbia, Manitoba, Ontario, Quebec, Nova Scotia and Newfoundland, but 383 cases were in Ontario. South-western Ontario was the primary focus of infection. There were 33 cases in Quebec and 24 in Manitoba, mainly found in and around Metropolitan Montreal and Winnipeg respectively.

     

Comparative Evaluation of 2 In-Clinic Assays for Vector-Borne Disease Testing in Dogs

Vector-borne agents comprise medically important infections affecting dogs throughout much of the world. Sensitive detection of antibodies directed at tick-borne disease-causing organisms in dogs is diagnostically important for veterinarians, pets and their owners, and epidemiologically important for public health surveillance. The SNAP 4Dx Plus Test (IDEXX Laboratories, Inc., Westbrook, ME) identifies antibodies to or infection with multiple tick-borne pathogens and canine heartworm antigen in a single assay. Recently, VetScan FLEX4 Rapid Test (Abaxis, Inc., Union City, CA) was launched as a new assay to detect tick-borne pathogen antibodies and heartworm antigen. In the present study, we evaluated the comparative performance of SNAP 4Dx Plus (SNAP) and FLEX4 Rapid Test (FLEX4) using samples selected based on geographic distributions for canine vector borne diseases, including Borrelia burgdorferi (n = 105), Anaplasma phagocytophilum (160), Anaplasma platys (115), Ehrlichia canis (154), Ehrlichia ewingii (163), Ehrlichia chaffeensis (151) and Dirofilaria immitis (105). Canine vector borne diseases infection status was established for each sample by a combination of reference methods that included necropsy (D. immitis, heartworm disease), Western immunoblotting (B. burgdorferi), immunofluorescence assays (A. phagocytophilum and E. canis) and species-specific ELISAs (A. platys, E. canis, E. ewingii and E. chaffeensis). For comparisons among the 2 assays, samples were evaluated per the manufacturers’ instructions for each test kit.By testing each same sample set compared to the defined reference results, sensitivities differed substantially between SNAP and FLEX4, at 95.5 vs. 40.9%, respectively for B. burgdorferi, 97.1% vs. 61.4% for E. canis, 98.2% vs. 59.3% for E. ewingii, 64.3% vs. 35.7% for E. chaffeensis, 84.5% vs. 12.7% for A. phagocytophilum, 83.3% vs. 33.3% for A. platys, and 94.1% vs. 88.2% for D. immitis. Specificities for both rapid assay tests ranged from 98% to 100%.Based upon the comparative results derived from this study, the SNAP test was more sensitive than the FLEX4 test for detection of antibodies to all tick-borne pathogens and heartworm disease (Dirofilaria immitis) antigen in dogs.     

D-dimer deposits in lungs and kidneys suggest its use as a marker in the clinical workup of dogs with heartworm (Dirofilaria immitis) disease

It has been reported that dogs with heartworm disease (Dirofilaria immitis) show increased plasma levels of D-dimer, a fibrin degradation product present in the blood after a blood clot is degraded by fibrinolysis. In the present study the authors show that, in dogs with both experimental and natural infections with D. immitis, D-dimer deposits in lungs and kidneys are associated with pulmonary thromboembolism and microfilariemic status, as well as there was a clear association between increased plasma values of D-dimer and positive staining in immunohistochemistry. Results suggest that the monitoring of D-dimer levels in infected dogs could be useful in evaluating the presence of pulmonary thromboembolism in the lungs and that microfilariae may induce microthrombosis in kidneys, thus contributing to renal pathology.     

Pulmonary artery dissection causing haemothorax in a cat: potential role of Dirofilaria immitis infection and literature review

A 7-year-old male castrated domestic short-haired cat suddenly died. Gross examination revealed severe right-sided haemothorax with blood clots, four adult filarial nematodes in the blood clots and the caudal vena cava and haemorrhage dissecting into the tunica media of the right pulmonary artery. Histopathological investigation showed fibrosis of the tunica intima and disorganization/fragmentation of the elastic fibres accompanied by fibrous tissue deposition in the tunica media of both branches of pulmonary artery. Degenerative vasculopathy (intimal fibromuscular hyperplasia and medial hypertrophy/hyperplasia) involving pulmonary arteries was also observed. The polymerase chain reaction amplification and sequencing confirmed the identification of the parasite as Dirofilaria immitis. A diagnosis of pulmonary artery dissection with haemothorax and concomitant heartworm disease was formulated. Degenerative processes of the tunica media have been reported to cause pulmonary artery dissection in both humans and animals. Pulmonary artery remodelling induced by heartworms may be considered the underlying cause in the first case of feline pulmonary artery dissection, herein described.     

Serological update and molecular characterization of Dirofilaria immitis in dogs,South Korea

Eighty-one dogs in the Chungnam province were tested for heartworm (Dirofilaria immitis) infection by ELISA (SNAP test, IDEXX Laboratories, Maine, USA). Seventeen (20.9%) of the 81 samples were found to be positive and further analyzed by 16S rRNA sequencing. In this study, all dogs tested lived outdoors. Using the χ² test and Fisher’s exact test, no significant differences in the prevalence of dirofilariasis were observed among different gender and age groups, although the prevalence of this disease among dogs 2–4 yrs of age remains highest. Sequence analysis revealed that the species prevalent in Chungnam province were genetically distinct from the type strain of D. immitis based on the nucleotide deletion found at position nt 276 (cytosine) and nucleotide substitution at position 428 (G to A) of the partial 16S rRNA sequence of the type strain. Furthermore, phylogenetic analysis suggests at least 2 groups of D. immitis circulating in the Chungnam area between the year 2007 and 2008.     

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.     

Molecular detection of filarial nematode parasites in Japanese black bears (Ursus thibetanus japonicus) from Iwate Prefecture, Japan

This study aimed to detect filarial parasites in blood samples of Japanese black bears (Ursus thibetanus japonicus) collected from Iwate Prefecture, Japan. Positive amplicons were obtained from 26 out of 30 samples by nested PCR targeting 18S ribosomal RNA gene and first internal transcribed spacer regions. DNA sequences of Mansonella sp. close to M. ozzardi and Dirofilaria sp. were detected for eight and 11 positive amplicons, respectively. Co-infection was detected for the remaining seven amplicons. Dirofilaria sp. was identified as D. ursi by further genetic analysis of 5S ribosomal RNA gene sequence. The results of this study will contribute to further investigations of Japanese black bears for monitoring their risk as a reservoir of possible zoonotic filarial parasites.     

Heartworm in dogs in Canada in 1986

In late December 1986, 1224 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1986; 46% of them responded. Veterinarians reported that 150,989 dogs were blood-tested for microfilariae and 869 dogs were found with heartworm. Another 65 dogs were amicrofilaremic but diagnosed with heartworm disease and one was found with heartworm at necropsy to give the total number diagnosed in 1986 as 935 (0.62%).

Heartworm was reported from Manitoba, New Brunswick, Ontario and Quebec, but most (810) of the cases were from Ontario. South-western Ontario continued to be the primary focus of the infection in Canada. There were 103 cases reported from Quebec, mostly from and around Montreal, and 21 cases from Manitoba, from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 33% of the cases were observed with clinical signs of heartworm disease and 81% had a history of not having left Canada.

     

Heartworm in dogs in Canada in 1985

In late December 1985, 1485 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1985 and 44% of them responded. Veterinarians reported that 137,300 dogs were blood-tested to check for microfilariae and 1210 dogs were found with heartworm. Another 36 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number diagnosed in 1985 as 1247 (0.91%).

Heartworm was reported from all provinces except Prince Edward Island, Newfoundland and Saskatchewan but most (1126) of the cases were in Ontario. Southwestern Ontario continued to be the primary focus of the infection in Canada. From Quebec, 91 cases were reported mostly from and around Montreal. From Manitoba, 19 cases were reported from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 28% of the cases were observed with clinical signs of heartworm disease and 78% had a history of not having left Canada.

     

Heartworm in dogs in Canada in 1984

In late December 1984, 1853 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1984 and 35% of them responded. Veterinarians reported that 97,794 dogs were blood-tested to check for microfilariae and 1417 dogs (1.45% of those tested) were found with heartworm. Another 34 dogs were amicrofilaremic, but were diagnosed as having heartworm disease, to give the total number diagnosed in 1984 as 1451 (1.48%). Heartworm was reported from all provinces except Prince Edward Island and Newfoundland but most (1310) of the cases were in Ontario. In Quebec, 126 cases were reported mostly from west of Montreal.

Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 27% of the cases were observed with clinical signs of heartworm disease and 72% had a history of not having left Canada. Southwestern Ontario continued to be the primary focus of the infection.

     

Molecular survey of Dirofilaria species in stray dogs,red foxes and golden jackals from Vojvodina,Serbia

Cardiopulmonary dirofilariosis in dogs and other carnivores is caused by Dirofilaria immitis, while Dirofilaria repens usually causes a subcutaneous infection. The importance of red foxes and golden jackals in the epidemiology of dirofilariosis remains unknown. Thus, the aim of this study was to conduct a cross-sectional molecular survey of Dirofilaria species in stray dogs, red foxes and golden jackals from the endemic region of Vojvodina, Serbia, in order to determine and update data on their prevalence and provide insight into the epidemiological importance of wild canids.A total of 59 blood samples from stray dogs, 94 from red foxes and 32 from golden jackals were collected and screened by real-time PCR targeting a 115-bp fragment of the mitochondrial 12S gene of filarioids and by conventional PCR assay targeting a 484–524-bp fragment of 5.8S-ITS2-28S locus of filarioids.The cross-sectional molecular survey detected the filarioid mitochondrial 12S gene fragment in stray dogs (27.1 %), red foxes (8.5 %) and golden jackals (6.3 %) in the same endemic region of Vojvodina, Serbia. Only D. immitis was detected in stray dogs, while both D. immitis and D. repens were detected in populations of red foxes and golden jackals. These results outline a possible interaction of D. immitis infection between the dog population and the wild canid populations, while D. repens was found to circulate mostly in golden jackals and red foxes populations.     

Absence of Wolbachia endobacteria in Sri Lankan isolates of the nematode parasite of animals Setaria digitata

Setaria digitata is an animal filarial parasite with natural hosts of cattle and buffaloes that causes mild disease conditions. Infection of non-permissive hosts such as goats, sheep and horses, by this nematode can cause cerebrospinal nematodiasis that leads to lumbar paralysis and the eventual death of the animals and inflicts considerable economic losses on livestock farmers. Wolbachia are obligate mutualistic endosymbionts for some filarial nematodes and are currently being targeted for the control of diseases caused by these parasites. However, little is known about the occurrence of this endosymbiont in the Setariidae family. In this work, worms collected from infected cattle in Sri Lanka were morphologically identified as S. digitata and tested for the presence of Wolbachia by PCR screening using the WSP- and Wolbachia-specific 16S rRNA and multilocus sequence typing primers that were designed to amplify the gatB, coxA, hcpA, ftsZ and fbpA sequences of Wolbachia. The presence of endobacteria in S. digitata was also examined by whole-mount immunofluorescence staining of the parasites and transmission electron microscopic studies. These analyses did not produce evidence of presence of Wolbachia or any other endosymbiotic bacteria in S. digitata, whereas such evidence was found in Brugia malayi, which was used as a positive control in this study.     

Subcutaneous and cardiopulmonary dirofilariasis in a dog

Canine subcutaneous dirofilariasis is rare and typically caused by Dirofilaria repens. An adult male, intact, mixed breed dog from South Carolina, USA, was examined because of physical trauma to the right maxilla. The dog died during hospitalization and was submitted for necropsy. Gross post-mortem changes included approximately 150 adult nematodes morphologically consistent with Dirofilaria spp. in the pulmonary artery, right atrium, and right ventricle. Histologically there was widespread proliferative pulmonary endarteritis with intraluminal nematodes morphologically consistent with Dirofilaria spp. Four similar nematodes were present in the subcutaneous tissue of the left medial thigh and tibial area. These nematodes were located within the fascia and skeletal muscles. They were surrounded by epithelioid macrophages and multinucleated giant cells, with some lymphocytes and plasma cells, and areas of mineralization (interpreted as mineralized cross sections of nematodes). Nematodes were morphologically identified as D. immitis. Subcutaneous dirofilariasis caused by D. immitis occurs when migrating nematode larvae develop into adults in the subcutaneous and skeletal muscle tissues.Key clinical message:Subcutaneous dirofilariasis in dogs is rare and most often associated with Dirofilaria repens. This case highlights the pathology findings of subcutaneous D. immitis in a dog.     

Aberrant heartworm migration to the abdominal aorta and systemic arteriolitis in a dog presenting with vomiting and hemorrhagic diarrhea

A 2-year-old Dachshund was presented for vomiting and diarrhea. Abdominal ultrasound revealed Dirofilaria immitis in the abdominal aorta and an avascular segment of small intestine. The dog was euthanized. Necropsy revealed D. immitis in the abdominal aorta and widespread necrotizing arteriolitis. This is a unique presentation of aberrant migration of D. immitis.     

Occurrence of novel and rare subtype families of Cryptosporidium in bamboo rats (Rhizomys sinensis) in China

This report is the first to describe Cryptosporidium infection in bamboo rats (Rhizomys sinensis). Ninety-two fresh fecal specimens were collected from a pet market in Ya’an City, China. One Cryptosporidium isolate from an asymptomatic host and two isolates from separate hosts with diarrhea were obtained by using Sheather's sucrose flotation technique and modified acid-fast staining. The Cryptosporidium spp. were genotyped by nested PCR and nucleotide sequencing of the small subunit rRNA (SSU rRNA), 70-kDa heat shock protein (HSP70), oocyst wall protein (COWP), and actin genes: isolates were identified as Cryptosporidium parvum with minor nucleotide differences at all four loci. Further subtyping was performed by PCR amplification and DNA sequence analysis of the 60-kDa glycoprotein (gp60) gene: two subtype families were detected, including a novel C. parvum subtype IIpA9 and a rare subtype IIoA13G1 (only reported in diarrheal patients of Sweden). Our results suggest that the bamboo rat is a reservoir host of C. parvum. Significantly, we discovered that the rare C. parvum subtype family IIo is also a zoonotic subtype and confirmed C. parvum subtype IIpA9 as a novel subtype family.     

Ethanolic extract of Azadirachta indica (A. Juss.) causing apoptosis by ROS upregulation in Dirofilaria immitis microfilaria

Dirofilaria immitis is the causative agent of cardiopulmonary dirofilariasis in the Canine family. The aim of the study was to evaluate the efficacy of the ethanolic extract of Azadirachta indica leaves (EEA) against the microfilaria (mf) of D. immitis in vitro. EEA was evaluated for different compound classes through HPTLC. Relative motility, mortality and morphological alterations were observed in the mf after exposure to EEA. The effect of EEA on redox status in the treated mf was evaluated by some key enzymatic and non-enzymatic parameters. An enhanced reactive oxygen species (ROS) level in the treated mf along with altered redox status was evident. DNA fragmentation and terminal-deoxynucleotidyl-transferase dUTP nick end labeling (TUNEL) confirmed apoptosis. In addition, western blotting revealed down-regulation of anti-apoptotic protein and up-regulation of pro-apoptotic proteins. Taken together, the microfilaricidal activity of EEA can be attributed to its capacity to inflict oxidative stress culminating in apoptosis.