Canine filarial infections in Tuscany, central Italy

This study was conducted in order to investigate the epidemiology of filarial species in a region of central Italy (Tuscany) in dogs that did not undergo prophylaxis for filariasis. From 2007 to 2009, 630 blood samples were collected from 40 kennels throughout the regional territory. Samples were analysed with Knott's modified test and with an enzyme-linked immunosorbent assay (ELISA) for the detection of Dirofilaria immitis antigens, those positive for microfilariae were also subjected to histochemical staining for acid phosphatase activity to validate the identification of the species. An overall elevated prevalence of filariasis (23.2%) was found. Dirofilaria immitis and D. repens were found to be the dominant species, with similar prevalences (12.5% and 12.1% respectively), while Acanthocheilonema reconditum was less common (1.9%). Results of this study indicate that monitoring for filariasis should be kept at a high level. A constant monitoring of the canine sanitary status should also be carried out for the protection of human health, considering the zoonotic potential of filarial worms.     

Survey of canine heartworm in the city of Recife, Pernambuco, Brazil.

Six hundred and eleven random-source dogs (338 male, 273 female) one year of age or older, from six sections of the city of Recife, Pernambuco, were examined antemortem for circulating microfilariae Dirofilaria immitis and Dipetalonema reconditum adult heartworm (D. immitis) antigen, and examined postmortem for adult heartworms. The prevalence of heartworm infection was 2.3% (14/611), as determined by necropsy for adult worms, and 1% (6/611) had circulating microfilariae of D. immitis; thus, 57.1% of the heartworm-infected dogs had occult infections. The results of serological testing indicated that 1.3% (8/611) of the dogs were positive for adult heartworm antigen. A total of 42 (6.9%) of the dogs had microfilariae of D. reconditum; 40 of these had only D. reconditum and two additional dogs had microfilariae of both species, D. immitis and D. reconditum.     

Onchocerca volvulus: molecular cloning, primary structure, and expression of a microfilarial surface-associated antigen.

Although the filarial nematode parasite Onchocerca volvulus is an important human pathogen in large areas of Africa and Latin America, little is known of the molecular interactions that govern the clinical status of patients with this chronic, debilitating disease. As a step toward defining the parasite molecules important to the immunobiology of host-parasite interactions, we have identified and cloned a major surface-associated antigen expressed by O. volvulus microfilariae. Radiolabeling experiments demonstrated that O. volvulus microfilariae have a limited repertoire of peptides at the surface. Prominent among these labeled peptides is an 18-kDa component. Immunological cross-reactivity between a surface-associated component of Dirofilaria immitis microfilariae and the 18-kDa surface-associated molecule from O. volvulus was exploited in a strategy to clone this potentially important O. volvulus microfilarial antigen. The cross-reacting antibodies were used to immunoscreen O. volvulus cDNA expression libraries. One clone, M2f.e, contained an open reading frame of 495 bp encoding an 18.1-kDa protein (OVMS18). Antibodies produced against the expression product of M2f.e recognized an 18-kDa component in extracts of O. volvulus microfilariae and bound to the surface of intact O. volvulus and D. immitis microfilariae. Southern blot analyses showed that M2f.e-like sequences are present in the genomic DNA of a number of filarial nematode species, but not in DNAs from nonfilarial nematode species.     

Preliminary results on the effect of tetracycline on the embryogenesis and symbiotic bacteria (Wolbachia) of Dirofilaria immitis. An update and discussion.

The distribution and phylogeny of Wolbachia in filarial species suggests that these endosymbiotic bacteria may be important in the biology of their filarial hosts. An experiment to falsify this hypothesis would be to treat filarial worms with antibiotics which are active against intracellular bacteria. Indeed, it has already been shown that tetracycline treatment inhibits development in a model filarial species (Brugia pahangi) at different stages of the life cycle, in both mosquito and mammalian hosts. Here we discuss these previous data and present new results on the effect of tetracycline on the embryogenesis of the canine filaria Dirofilaria immitis.     

First finding of Dirofilaria repens in a natural population of Aedes albopictus

The invasive mosquito Aedes albopictus (Skuse) (Diptera: Culicidae) has become widespread in Italy during the past decade. Also Italy has foci of canine filariasis caused by Dirofilaria (Spirurida: Onchocercidae), due to subcutaneous D. repens Railliet & Henry as well as the dog heartworm D. immitis (Leidy) transmitted by various vector mosquitoes (Diptera: Culicidae). In 2002, at Fiumicino, west of Rome (Lazio Region), 17% of dogs were found to have D. repens microfilariae in peripheral blood. To evaluate the role of Ae. albopictus as a vector of Dirofilaria in this area, female mosquitoes were collected daily, June-October 2002, landing on dog or human bait in a rural house at Focene. Mosquitoes were maintained at 27 degrees C and 70% RH for 6 days, to allow development or purging of filaria larvae, then identified and frozen for subsequent molecular assay with filaria-specific ribosomal S2-S16 primers. To distinguish specimens harbouring infective L3 Dirofilaria larvae, DNA was extracted separately from the mosquito abdomen and head-thorax. Dirofilaria species were identified by sequencing, confirmed by polymerase chain reaction of positive specimens using primers specific for D. immitis and D. repens. Dirofilaria DNA was detected in 3/154 (2%) of Ae. albopictus females examined: D. repens DNA in head-thorax and abdomen of one collected 27th July; D. immitis in the abdomen of one collected 24th September; DNA of both D. immitis and D. repens in the head-thorax of one collected 11th October 2002. Thus Ae. albopictus is a potential vector of both Dirofilarias in Italy, representing risks for veterinary and human health.     

Genetic diversity and fitness in black-footed ferrets before and during a bottleneck

The black-footed ferret (Mustela nigripes) is an endangered North American carnivore that underwent a well-documented population bottleneck in the mid-1980s. To better understand the effects of a bottleneck on a free-ranging carnivore population, we used 24 microsatellite loci to compare genetic diversity before versus during the bottleneck, and compare the last wild population to two historical populations. We also compared genetic diversity in black-footed ferrets to that of two sibling species, the steppe polecat (Mustela eversmanni) and the European polecat (Mustela putorius). Black-footed ferrets during the bottleneck had less genetic diversity than steppe polecats. The three black-footed ferret populations were well differentiated (F(ST) = 0.57 +/- 0.15; mean +/- SE). We attributed the decrease in genetic diversity in black-footed ferrets to localized extinction of these genetically distinct subpopulations and to the bottleneck in the surviving subpopulation. Although genetic diversity decreased, female fecundity and juvenile survival were not affected by the population bottleneck.     

Helminths of the ocelot from southern Texas

In the USA, the ocelot (Leopardus pardalis) is a highly endangered felid found only in a few remaining vestiges of native thornshrub brushland in the Lower Rio Grande Valley (LRGV) of extreme southern Texas. From 1987-1998, carcasses of 15 adult ocelots that died of vehicular accidents or natural causes were examined for helminths. All cats had 1-8 (mean = 3) helminth species. All were infected with 1-101 (mean +/- SE = 32 +/- 7) Toxascaris leonina. Other helminths from these ocelots were Alaria marcianae, Brachylaima sp., Mesocestoides lineatus, Taenia rileyi, Oncicola canis, Dirofilaria immitis, Physaloptera rara, Ancylostoma tubaeformae, Cylicospirura chevreuxi, Vogeloides felis, and Metathelazia californica. Additionally, two cats had scarring of the aorta with lesions typical of those caused by Spriocerca lupi, although larval nematodes were not seen. A clinal variation in size of nearly three orders of magnitude was noted in the diplostomatid trematodes in the small intestine of one adult male ocelot. Despite the differences in size, all specimens appeared morphologically identical and were regarded as A. marcianae. Helminth prevalences and abundances, including those of potentially pathogenic species like D. immitis, were low. Although a single heartworm infection may have contributed to the death of one ocelot, helminth infections in general seemed to be of no great consequence to this endangered ocelot population. The helminth fauna of ocelots in the LRGV is reflective of that from wild felids in general; all have been reported previously from the bobcat (Lynx rufus) and mountain lion (Puma concolor) elsewhere in Texas.     

Phylogeny of Wolbachia in filarial nematodes.

Intracellular bacteria have been observed in various species of filarial nematodes (family Onchocercidae). The intracellular bacterium of the canine filaria Dirofilaria immitis has been shown to be closely related to Wolbachia, a rickettsia-like micro-organism that is widespread among arthropods. However, the relationships between endosymbionts of different filariae, and between these and the arthropod wolbachiae, appear not to have been studied. To address these issues we have examined ten species of filarial nematodes for the presence of Wolbachia. For nine species, all samples examined were PCR positive using primers specific for the ftsZ gene of Wolbachia. For one species, the examined samples were PCR negative. Sequences of the amplified ftsZ gene fragments of filarial wolbachiae fall into two clusters (C and D), which are distinct from the A and B clusters recognized for arthropod wolbachiae. These four lineages (A-D) are related in a star-like phylogeny, with higher nucleotide divergence observed between C and D wolbachiae than that observed between A and B wolbachiae. In addition, within each of the two lineages of filarial wolbachiae, the phylogeny of the symbionts is consistent with the host phylogeny. Thus, there is no evidence for recent Wolbachia transmission between arthropods and nematodes. Endosymbiont 16S ribosomal DNA sequences from a subset of filarial species support these findings.     

Combined ivermectin and doxycycline treatment has microfilaricidal and adulticidal activity against Dirofilaria immitis in experimentally infected dogs

There is still a pressing need for effective adulticide treatment for human and animal filarial infections. Like many filarial nematodes, Dirofilaria immitis, the causative agent of canine heartworm disease, harbours the bacterial endosymbiont Wolbachia, which has been shown to be essential for worm development, fecundity and survival. Here the authors report the effect of different treatment regimens in dogs experimentally infected with adult D. immitis on microfilariemia, antigenemia, worm recovery and Wolbachia content. Treatment with ivermectin (IVM; 6 microg/kg per os weekly) combined with doxycycline (DOXY; 10 mg/kg/day orally from Weeks 0-6, 10-12, 16-18, 22-26 and 28-34) resulted in a significantly faster decrease of circulating microfilariae and higher adulticidal activity compared with either IVM or DOXY alone. Quantitative PCR analysis of ftsZ (Wolbachia DNA) and 18S rDNA (nematode DNA) absolute copy numbers showed significant decreases in Wolbachia content compared with controls in worms recovered from DOXY-treated dogs that were not, however, associated with worm death. Worms from IVM/DOXY-treated dogs, on the other hand, had Wolbachia/nematode DNA ratios similar to those of control worms, suggesting a loss of both Wolbachia and nematode DNA as indicated by absolute copy number values. Histology and transmission electron microscopy of worms recovered from the IVM/DOXY combination group showed complete loss of uterine content in females and immunohistochemistry for Wolbachia was negative. Results indicate that the combination of these two drugs causes adult worm death. This could have important implications for control of human and animal filarial infections.     

Tunga penetrans: molecular identification of Wolbachia endobacteria and their recognition by antibodies against proteins of endobacteria from filarial parasites

In search of Wolbachia in human parasites, Wolbachia were identified in the sand flea Tunga penetrans. PCR and DNA sequencing of the bacterial 16S rDNA, the ftsZ cell division protein, the Wolbachia surface protein (wsp) and the Wolbachia aspartate aminotransferase genes revealed a high similarity to the respective sequences of endosymbionts of filarial nematodes. Using these sequences a phylogenetic tree was generated, that indicates a close relationship between Wolbachia from T. penetrans and from filarial parasites, but possibly as a member of a new supergroup. Ultrastructural studies showed that Wolbachia are abundant in the ovaries of neosomic fleas, whereas other, smaller and morphologically distinct, bacteria were observed in the lumen of the intestine. Wolbachia were labeled by immunohistology and immunogold electron microscopy using polyclonal antibodies against wsp of Drosophila, of the filarial parasite Dirofilaria immitis, or against hsp 60 from Yersinia enterocolitica. These results show that as in filariasis, humans with tungiasis are exposed to Wolbachia. Furthermore, antisera raised against proteins of Wolbachia from arthropods or from filarial parasites can be immunologically cross-reactive.     

The detection of antibodies in human and animal filariases by counterimmunoelectrophoresis with Dirofilaria immitis antigens.

Counterimmunoelectrophoresis revealed the presence of precipitin antibody in all of 6 dogs and the 1 cat infected with Dirofilaria immitis and in the serum of 17 of 24 individuals living in a setting of hyperendemic subperiodic bancroftian filariasis. Antigens used in the test were prepared from microfilariae and adult male D. immitis. Some humans and animals had antibodies to both antigens while others had antibodies against microfilariae or adult worms only. The presence of soluble circulating antigen was detected in the sera of two dogs with high microfilaraemias.     

Population Indices Versus Correlated Density Estimates of Black-Footed Ferret Abundance

ABSTRACT Estimating abundance of carnivore populations is problematic because individuals typically are elusive, nocturnal, and dispersed across the landscape. Rare or endangered carnivore populations are even more difficult to estimate because of small sample sizes. Considering behavioral ecology of the target species can drastically improve survey efficiency and effectiveness. Previously, abundance of the black-footed ferret (Mustela nigripes) was monitored by spotlighting and generating indices of relative abundance because reintroduced populations were slow to establish. Indices, however, lack variance estimates and are costly to generate for the black-footed ferret. We therefore used spotlight surveys and live-trapping in conjunction with a robust mark-recapture estimator to improve abundance monitoring for the black-footed ferret, one of North America's most endangered carnivores. We estimated abundance of the black-footed ferret at Shirley Basin, Wyoming, USA, using correlated density estimates and Program MARK. We compared our results to 2 indices of relative abundance, minimum number alive and predicted number of ferrets from litter counts. The correlated density estimate for the black-footed ferret (N̂_R = 229; 95% CI = 161–298) was similar to minimum number alive (N̂_R = 192) and predicted number of ferrets from litter counts (N̂_R= 235). The efficiency and effectiveness of survey methods we used for the black-footed ferret were high by carnivoran standards. Our results suggest that the sampling approach we utilized can be implemented for a fraction of the cost and effort required to generate 2 indices of relative abundance for the black-footed ferret. Although we recommend managers implement a similar survey approach to monitor abundance of reintroduced populations of the black-footed ferret, analysis with sparse data sets will be problematic. Until the black-footed ferret becomes widespread and abundant at a reintroduction site, spotlighting will remain preferable as a means to generate indices of distribution and relative abundance for the black-footed ferret.     

Immunodiagnosis of bancroftian filariasis—Problems and progress

The immunodiagnosis of bancroftian filariasis is a major challenge to the immunoparasitologist. Significant progress is yet to be made in developing convenient laboratory animal model and inin vitro cultivation of filarial parasites making it very difficult to obtain required amount of parasite material for research. Parasitological examination techniques are not useful in low microfilaraemia, occult or chronic.filarial infections. A precise and accurate immunodiagnostic technique is very much needed for successful filaria control programmes. Such a test will also avoid the need for laborious night blood examination in bancroftian filariasis. Due to comparatively easy availability, a good amount of work has been done to explore immunodiagnostic potential of heterologous filarial antigens isolated fromLitomosoide carinii, Dirofilaria immitis, Brugia malayi, Setaria digitata, Setaria cervi and number of other filarial species. However, there has been limited or no significant success due to number of false negative and false positive reactions. Extensive study has also been made with antigens isolated fromWuchereria bancrofti microfilariae. Soluble antigens of microfilariae have been used in different immunological techniques such as skin test, counter immuno electrophoresis, indirect haemagglutination test, indirect fluorescent antibody test and enzyme linked immunosorbent assay. Fractionation of Wuchereria bancrofti microfilarial soluble antigens yielded mfS3e antigen fraction which was found to be highly reactive in microfilaraemia by enzyme linked immunosorbent assay, but the yield of the purified antigen was not sufficient enough to make it a practical proposition for large scale isolation of antigen. Wuchereria bancrofti microfilarial excretory-secretory antigens were found to be specific and highly sensitive requiring as little as 0.35 ng antigen protein per well in penicillinase enzyme linked immunosorbent assay for detection of filarial antibody. One ml of culture fluid was found to be sufficient for 400,000 tests. Field evaluation of this test showed that it can replace laborious night blood examination. Assay systems have been developed for detection of filarial antigen in serum, urine, hydrocele fluid and immune complexes using immunoglobulins from chronic filarial sera and antisera to excretory filarial antigens. Further purification of excretory-secretory antigens by affinity chromatography and production of monoclonal antibodies should hopefully give suitable reagents for use in sensitive assays such as enzyme immuno assay and immuno radiometric assay, providing an ideal assay system for detection of active filarial infection in the not too distant future.     

Selective up-regulation of endothelial cell class I MHC expression by cytokines from patients with lymphatic filariasis

Local host immune responses to the lymphatic-dwelling filarial parasite Wuchereria bancrofti are important in the pathogenesis of the lymphangitis that leads to filarial elephantiasis. That the lymphatic endothelial cells may be important in this inflammatory process was shown by the ability of supernatants generated from filarial Ag-driven PBMC of individuals with filarial elephantiasis caused by W. bancrofti infection to up-regulate class I MHC expression on human umbilical vein endothelial cells when compared to unstimulated control supernatants from the same individual (relative fluorescence intensity = 159% +/- 13.5; p less than 0.001). In contrast, individuals with the same filarial infection but manifesting no lymphatic disease were unable to generate, in response to filarial Ag the cytokines required for this activation (relative fluorescence intensity = 93% +/- 2.6). Supernatants induced by a non-filarial Ag (purified protein derivative) were able to effect class I MHC up-regulation in both patient groups. The same filarial Ag-driven supernatants did not cause detectable class II MHC staining on human umbilical vein endothelial cells. These results suggest a likely role for parasite Ag-driven, cytokine-mediated endothelial cell activation in the pathogenesis of lymphatic inflammatory/obstructive filarial disease.     

Sperm viability in the black-footed ferret (Mustela nigripes) is influenced by seminal and medium osmolality

Fundamental knowledge of spermatozoa cryobiology can assist with optimizing cryopreservation protocols needed for genetic management of the endangered black-footed ferret. Objectives were to characterize semen osmolality and assess the influence of two media at various osmolalities on sperm viability. We examined the influence of Ham's F10 +Hepes medium (H) at 270, 400, 500 or 700 mOsm (adjusted with sucrose, a nonpermeating cryoprotectant) and TEST Yolk Buffer (TYB) with 0% (300 mOsm) versus 4% (900 mOsm) glycerol (a permeating cryoprotectant). Electroejaculates (n=16) were assessed for osmolality using a vapor pressure osmometer. For media comparison, semen (n=5) was collected in TYB 0%, split into six aliquots, and diluted in H270, H400, H500, H700, and TYB 0% or TYB 4%. Each sample was centrifuged (300 g, 8 min), resuspended in respective medium, and maintained at 37 degrees C for 3h. Sperm motility and forward progression were monitored every 30 min for 3h post-washing. Acrosomal integrity was monitored at 0 and 60 min post-washing. Results demonstrated that black-footed ferret semen has a comparatively high osmolality (mean+/-SEM, 513.1+/-32.6 mOsm; range, 366-791 mOsm). Ferret spermatozoa were sensitive to hyperosmotic stress. Specifically, sperm motility was more susceptible (P<0.01) to hyperosmotic conditions than acrosomal integrity, and neither were influenced (P>0.05) by hypotonic solutions. Exposure to TYB 4% glycerol retained more (P<0.01) sperm motility than a hyperosmotic Ham's (700 mOsm). These findings will guide the eventual development of assisted breeding with cryopreserved sperm contributing to genetic management of this rare species.     

Serological and molecular studies on Dirofilaria immitis in dogs from Turkey

We estimated the prevalence of Dirofilaria immitis infection in domestic dogs in five Turkish provinces - Sakarya, Kocaeli, Ankara, Elazig and Mersin - using a commercial ELISA kit for detecting circulating antigen and a PCR test for detecting circulating microfilarial DNA. A total of 211 whole-blood and serum samples were collected from dogs of various breeds, ages and life status (owned or stray). Sample population characteristics were recorded and examined for differences in prevalence. Additionally, we collected 15 blood samples from cats (14 owned and 1 stray) from Ankara province and used PCR to detect D. immitis infection. Twenty-seven (12.8%) of 211 dog samples were positive for D. immitis antigen by ELISA. No differences in prevalence were observed by sex (female: 14.4%; male: 10.7%; P>0.05). The prevalence of D. immitis infection varied with age: 11.8% in younger dogs (0.5-2 years) and 17.5% in older dogs (3-5 years). Prevalence between stray dogs (15.2%) and owned dogs (9.3%) did not differ (P>0.05). Prevalence rates were highest in Kocaeli province (18.3%), followed by Ankara (14.8%), Sakarya (12.3%) and Mersin (10.5%) provinces. Prevalence in Elazig province was 0%. No dogs or cats had microfilarial DNA detectable by PCR.     

Stick enzyme-linked immunosorbent assay using the avidin-biotin system for detection of circulating antigen in bancroftian filariasis

Detection of filarial antigen in different groups of sera was carried out by sandwich as well as inhibition enzyme-linked immunosorbent assays using antibody-coated sticks. Both systems were found to be equally sensitive in detecting antigen in 90% of microfilariae carriers. Incorporation of avidin-biotin in the sandwich assay system increased the sensitivity of antigen detection from 10⁻⁶ to 10⁻¹⁶ pg. A 67% decrease in the number of false negative results was observed when the sensitive avidin-biotin inhibition enzyme-linked immunosorbent assay system was used for analysis of filaria blood samples.