A prevalence survey and risk analysis of filariosis in dogs from the Mt. Vesuvius area of southern Italy.

A dog microfilariae prevalence and risk factor survey was conducted in 51 contiguous municipalities of the Mt. Vesuvius area (Campania region, southern Italy) in order to add data to the limited epidemiological information available regarding filarial worms in this zone. Between May 1999 and June 2000, blood samples were collected from 351 asymptomatic dogs. Blood samples were examined using a modified Knott's technique and histochemical staining in order to count and identify microfilariae. The results were subjected to statistical analysis and choroplethic municipal maps (MMs) were drawn by a geographical information system (GIS) software. Microfilariae were detected in 63 of the 351 dogs surveyed, constituting a total filarial prevalence of 17.9%. In particular, 56 dogs (15.9%) showed only microfilariae of Dipetalonema reconditum; three dogs (0.8%) only microfilariae of Dirofilaria repens; two dogs (0.6%) microfilariae of both D. reconditum and D. repens and two dogs (0.6%) microfilariae of both Dirofilaria immitis and D. repens. High D. reconditum prevalence was associated with hunting practice, masculine gender and older dogs. There was also a tendency to find high prevalence in dogs sampled in the afternoon.In conclusion, the presence of microfilariae of D. reconditum in 92% of microfilaraemic dogs indicates that this filarial worm was the predominant filarial species in dogs in the Mt. Vesuvius area.In addition, the general trends of the MMs showed that D. immitis and D. repens were present only in a few municipalities, whereas D. reconditum was widely and homogeneously spread throughout the entire study area.     

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.     

Rhipicephalus sanguineus (Ixodida, Ixodidae) as intermediate host of a canine neglected filarial species with dermal microfilariae

The life cycles of filarioids of dogs presenting dermal microfilariae have been little studied. Following the recent retrieval of dermal microfilariae identified as Cercopithifilaria sp. in a dog from Sicily (Italy), this study was designed to assess the role of the brown dog tick Rhipicephalus sanguineus as an intermediate host of this filarial species. An experimental tick infestation was performed on an infected dog using 300 nymphs of R. sanguineus. Engorged nymphs were collected and examined by both microscopic dissection and molecular analysis at five time points (i.e., the same day of tick detachment and 10, 20, 30 and 50 days post-detachment) to detect the presence and developmental stage of filariae in the ticks. A total of 270 engorged nymphs were collected from the dog and developing filarioid larvae detected in 10 (5%) out of 200 ticks dissected. Infective third-stage larvae were observed in 4 (2%) of the all dissected ticks, 30 days post-detachment. Twelve (6.6%) out of 181 samples molecularly tested were positive for Cercopithifilaria sp. This study demonstrates that nymphs of R. sanguineus feeding on a dog naturally infected by Cercopithifilaria sp. can ingest microfilariae, which develop up to the third infective stage thus suggesting that this tick species might act as an intermediate host of this little known canine filarioid.     

Dogs with patent Dirofilaria immitis infection have higher expression of circulating IL-4, IL-10 and iNOS mRNA than those with occult infection

Dirofilaria immitis is the agent of canine heartworm disease, in which adult worms reside in the pulmonary arteries, producing first stage larvae (microfilariae) that are released into the bloodstream. The present work describes the cytokine and iNOS mRNA expression in the peripheral blood of naturally infected dogs classified as either microfilariemic or amicrofilariemic. Results show that microfilariemic dogs had higher expression of IL-4 and iNOS mRNA than amicrofilariemic dogs. Furthermore, IL-10 mRNA expression was strongly expressed in dogs with circulating microfilariae, compared to only negligible expression in amicrofilariemic dogs. Finally, mf+ status was associated with a predominance in IgG1 production against worm antigens. These results would suggest that circulating mf may stimulate, like in other filarial infections, an immune bias towards unresponsiveness in D. immitis-infected dogs, consenting long-term adult worm survival.     

Morphological and molecular data on the dermal microfilariae of a species of Cercopithifilaria from a dog in Sicily

Dermal microfilariae found in a dog from Sicily, Italy, were characterized morphologically and genetically and differentiated from those of all the other blood microfilariae commonly found in dogs. In particular, the microfilariae were short (mean length of 186.7 μm), presented a body flattened dorso-ventrally and a rounded head, bearing a tiny cephalic hook. The genetic identity of microfilariae herein studied was also assessed by molecular amplification, sequencing and analyzing of multiple ribosomal ITS-2 and mitochondrial (cox1 and 12S) target genes. Both morphologic and genetic characterization as well as the molecular phylogenetic history inferred using sequences of a barcoding dataset were concordant in supporting the identification of Cercopithifilaria at the genus level. Surprisingly, microfilariae here examined were well distinct from Cercopithifilaria grassii (Noè, 1907), from northern Italy, and resembled those of a species described in Brazil, Cercopithifilaria bainae Almeida & Vicente, 1984. This paper provides evidence for the existence of a Cercopithifilaria species infesting a dog from Sicily and also presents a PCR protocol on skin samples as a tool for further epidemiological studies, which could provide evidence on the aetiology and the natural history of this filarial species.     

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%.     

Immune response to and tissue localization of the Wolbachia surface protein (WSP) in dogs with natural heartworm (Dirofilaria immitis) infection

Human and animal parasitic filarial nematodes, including the agent of canine and feline heartworm disease Dirofilaria immitis, harbour intracellular bacteria of the genus Wolbachia (Rickettsiaies). It is thought that these bacteria play an important role in the pathogenesis and immune response to filarial infection. Immunoglobulin G (total IgG, IgG1, IgG2) production against and immunohistochemical staining of tissues for the Wolbachia surface protein (WSP) from dogs with natural heartworm infection were evaluated. All infected dogs had significant total anti-WSP IgG levels compared to healthy controls. Interestingly, WSP was recognized by the IgG2 subclass in both microfilariemic dogs and in dogs with no circulating microfilariae (occult infection). However, microfilariemic dogs also produced gG1 antibodies. Positive staining for WSP was observed in lungs, liver and kidneys, in particular in glomerular capillaries of naturally infected dogs who had died from heartworm disease. Our results show for the first time that Wolbachia is recognized specifically by D. immitis--infected dogs and that the bacteria is released into host tissue. Furthermore, microfilariemic status appears to effect immune responses to this endosymbiont.     

Occurrence of filaria in domestic dogs of Samburu pastoralists in Northern Kenya and its associations with canine distemper

Samples of blood (serum, smears and blood preserved with ethanol) were collected from dogs during a vaccination campaign in northern Kenya in the years 2006 and 2007. Blood was screened for filarial parasites using molecular and microscopy methods and sera were tested for antibodies against canine distemper virus (CDV). Parasitological examination revealed the presence of two species of canine filariae: Acanthocheilonema dracunculoides and A. reconditum. The DNA from the former species was detected in 58% dogs sampled in 2006 and 36% dogs sampled in 2007, whereas the latter was found only in 4.2% samples collected in 2007. Microfilariae were found in 33.8% blood smears collected in 2006 and 10.6% blood smears collected in 2007. The seroprevalence of CDV was 33.4% in 2006 and 11.2% in 2007. The effect of sex, age and CDV-seropositivity/seronegativity on the occurrence of A. dracunculoides was evaluated. Infection by A. dracunculoides was more common in males and in dogs with a positive antibody titer for canine distemper, but evenly distributed among different age groups. The difference in the prevalence of A. dracunculoides in two isolated mountain ranges was not statistically significant. Methodologies available for detection and determination of canine filariae are compared, underlining methodical pitfalls arising through the determination of less common filarial species. The role of single epidemiological factors and possible association between canine distemper and filariasis 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.     

A simple molecular method for discriminating common filarial nematodes of dogs (Canis familiaris)

Accurate diagnosis of canine filariosis is essential for choosing correct therapeutic approach. Therefore, reliable methods for discriminating among the different filarial infections in dogs are needed. The authors report simple and highly specific molecular methods that identify the three most common filarial nematodes of European dogs: Dirofilaria immitis, D. repens and Acanthocheilonema (syn. Dipetalonema) reconditum, based on (1) PCR amplifications of mitochondrial DNA (12S rDNA and coxI) with general filarial primers followed by digestion with restriction enzymes that generates band polymorphisms clearly discriminating the three species and (2) PCR amplifications with species-specific primers to support the restriction analysis, in particular in the case of multiple infections.     

Ocular onchocercosis in dogs: aberrant infection in an accidental host or lupi onchocercosis?

Four adult dogs that had spent their entire life in Hungary, were found to be infected with filaroid nematodes of the genus Onchocerca. The morphology and location of the parasites as well as pathological lesions were similar to those described earlier in the one Hungarian and five US dogs. Only moderate morphological differences were noted between the adults of Onchocerca sp. infecting dogs and O. volvulus of man or O. lienalis of cattle. Nevertheless, the morphology of microfilariae of Onchocerca from dogs is unique within the genus. Their length was less than half the length of microfilariae of other Onchocerca spp. known so far. In addition to size differences, several characteristic morphological features were observed. The unsuccessful attempt to infect dogs with O. lienalis, the absence of O. volvulus and O. lienalis in endemic regions of canine onchocercosis, the different size, morphology, and location of the adults in dogs and cattle, the exceptionally small size and unique morphology of microfilariae of Onchocerca of canids indicate that a distinct species might be responsible for canine onchocercosis. Since the larval concentration in the skin was high (50-3600 microfilariae g(-1)) in all affected dogs, the diagnosis prior to surgical removal of worm nodules can be based on the examination of a small skin snip collected from the head or abdominal region. Infections in dogs may provide a model to study human onchocercosis, therefore, further studies are encouraged on the feasibility of experimental infection of dogs with this Onchocerca species.     

Helminth parasites of dogs from two resource-limited communities in South Africa

Biological samples were collected from dogs in resource-limited communities in the North-West and Gauteng Provinces of South Africa to assess the prevalence of helminth parasitism. These samples included adhesive tape peri-anal skin swabs and fresh faecal samples for helminth examination, and thick and thin blood films (smears) and whole-blood samples in anticoagulant for examination of filarial nematode microfilariae and haemoprotozoa. The eggs of Ancylostoma caninum, Toxocara canis, Toxascaris leonina, Dipylidium caninum and taeniids were identified. None of the blood samples and smears tested positive for microfilariae of Dirofilaria immitis or Dipetalonema spp. or for haemoprotozoa. The adhesive tape swabs were negative for cestode eggs and segments. Most of the helminth parasites identified in this study are zoonotic and consequently are regarded as a public health hazard.     

Identification of microfilaria in a mammary mass aspirate from a female dog

A filarial nematode larva (microfilaria) was found in an aspirate of a mammary mass from a dog imported to Saskatchewan from Myanmar. This was an unusual location for a microfilaria and numerous filarial nematodes infecting dogs in Asia had to be considered. This report describes the laboratory diagnosis of canine heartworm disease in this dog.     

Preliminary findings on the efficacy of selamectin in the treatment of dogs naturally infected with Dirofilaria repens

Subcutaneous dirofilariosis caused by Dirofilaria repens is common in dogs and it is an emerging helminthozoonosis in Europe, Asia, Africa and also in Hungary. Macrocyclic lactones are used for preventing the infection; however, their activity against the microfilariae and mature stages of this species is questionable. Selamectin is widely used for the prophylaxis of heartworm (D. immitis) infection. The objective of the present study was to test the microfilaricidal efficacy of the topical formulation of selamectin in dogs naturally infected with D. repens . A total of 78 Beagle dogs were examined for the presence of circulating microfilariae by Knott's test. Twenty-three of the microfilaraemic dogs were divided into four groups and included in the trial. The dogs received monthly or biweekly selamectin treatment and were subjected to monthly blood testing for a period of 252 or 336 days. At the end of the study, 65% of the dogs were not microfilaraemic and the rest had low number of microfilariae in their blood. These results indicate that chronic spot-on selamectin treatment may be a useful tool also in the control of canine subcutaneous dirofilariosis.     

Dirofilaria repens—An etiological factor or an incidental finding in cytologic and histopathologic biopsies from dogs

Dirofilaria repens is an endemic, zoonotic parasite of carnivores, causing subcutaneous dirofilariasis, which is mostly asymptomatic. The aim of this study was to describe 22 cases of canine subcutaneous dirofilariasis. The cytologic and histopathologic samples were collected from dogs, which presented with various clinical signs such as cutaneous/subcutaneous nodules, hydropericardium, ascites, and lymphadenomegaly. The cytologic or histopathologic examination revealed purulent, pyogranulomatous, granulomatous or eosinophilic dermatitis/panniculitis, and the presence of D repens microfilariae or adults. The microfilariae or adults were also found incidentally in neoplastic cutaneous or subcutaneous tumors and in a sialocele. For the first time, microfilariae were also detected and described in pericardial and abdominal effusions and in enlarged reactive lymph nodes. Although it is well known that D repens can cause dermatitis and panniculitis in dogs, no previous reports of D repens microfilariae in body cavity fluids were found. The role of this parasite in the accumulation of body cavity fluid or in reactive lymphadenomegaly requires further investigation. Due to its zoonotic potential, and apparently underestimated pathogenicity, each case of canine subcutaneous dirofilariasis should be treated.     

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.

     

Onchocerca gutturosa and Onchocerca lienalis in cattle: variation in length of microfilariae by site of recovery

Dermal microfilariae recovered from specimens obtained from umbilical and cervical sites of cattle infected with adult Onchocerca gutturosa alone or with adults of O gutturosa and O lienalis were measured and compared with uterine microfilariae obtained directly from gravid female worms of each species. Uterine microfilariae of O gutturosa were longer than dermal microfilariae obtained from cattle harboring only adults of O gutturosa. Dermal microfilariae were recovered from umbilical and cervical sites in these cattle. Those found at the cervical site had lengths equal to or greater than lengths of microfilariae recovered from the umbilical site. There was a significant (P less than 0.0001) shift in length across populations of microfilariae of O gutturosa from various sites in its bovine host, with a progressive decrease in length between microfilariae recovered from the worm's uterus, microfilariae from the cervical dermis, and microfilariae from the umbilical dermis, respectively. A similar direct comparison was not possible for microfilariae of O lienalis, because none of the cattle was infected with only adult worms of this species. In an indirect comparison, microfilariae of O lienalis were identified at the umbilicus, but their presence in the cervical region could not be determined unequivocally because of confounding of microfilariae length by concurrent infection with O gutturosa. Uterine microfilariae from O lienalis were longer than uterine microfilariae of O gutturosa, although a degree of overlap in the range of measurements existed between species.     

Histochemical differentiation of Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema dracunculoides microfilariae by staining with a commercial kit, Leucognost-SP.

The diagnosis of canine heartworm infection is based upon the presence of circulating Dirofilaria immitis microfilariae or on techniques for the detection of serum antibodies or antigens. In the first of these, discrimination between D. immitis, D. repens and Acanthocheilonema dracunculoides microfilariae is based upon the acid phosphatase histochemical stain. In this paper, we propose an alternative technique for histochemical staining using a commercial kit test of naphthol-AS-OL (Leucognost-SP). This offers the advantages of speed and simplicity as compared to the standard Barka procedure.     

Renal haemosiderosis in association with canine heartworm disease

Twenty-five cases of renal haemosiderosis associated with canine heartworm disease are reported. Severe intravascular haemolysis was a feature of six dogs with filariae in the right atrium and venae cavae (caval syndrome). An association between haemosiderin deposition and filarial burden was apparent in nineteen dogs not showing clinical signs of caval syndrome. The possible reasons for the sub-clinical haemolysis in canine heartworm disease are discussed with regard to current literature.