Dipetalonema graciliformis (Freitas, 1964) from the red-handed tamarins (Saguinus midas, Linnaeus, 1758) in French Guiana.

Six Dipetalonema species have been reported from Neotropical monkeys, Dipetalonema gracile, Dipetalonema graciliformis and Dipetalonema caudispina being the dominant species found in French Guiana primates. Adult filarioids isolated from the abdominal cavity of tamarins (Saguinus midas) in French Guiana were morphologically and molecularly identified as D. graciliformis. Phylogenetic analysis based on DNA and amino acid sequences of the cox1 gene as well as the concatenated sequences of the cox1 and the 18S genes indicated that D. graciliformis belongs to the clade 4 (ONC4) of Onchocercidae. Blast analysis of the 18S rDNA revealed that D. graciliformis in the studied tamarins is conspecific with the filarioid circulating in howler monkeys (Alouatta macconnelli) in French Guiana, previously referred to as unidentified Onchocercidae species.

Co-infection with filarial nematodes in Sapajus macrocephalus and Cebus albifrons (Primates: Cebidae) from the Peruvian Amazon.

Dipetalonema caudispina (Molin, 1858) and D. gracile (Rudolphi, 1809) (Filarioidea: Onchocercidae) are two of six known species of filarial nematodes that parasitize Neotropical non-human primates. Adult filariae were collected from the thoracic and abdominal cavities of 38 of 44 specimens of Sapajus macrocephalus (Spix, 1823) and nine of ten specimens of Cebus albifrons (Humboldt, 1812) (Primates: Cebidae), distributed in the Yavarí-Mirín river basin and used locally for human consumption. Co-occurrence of D. caudispina and D. gracile is reported for the first time, with a prevalence of 18.5% (10 of 54 hosts examined). Our finding of D. caudispina and D. gracile in cebids from the Peruvian Amazon constitutes a new geographical record for both filariae, two new host records for D. caudispina, and the first report of D. gracile in S. macrocephalus. In addition, we provide morphometric data for D. caudispina, complementing the original description, as well as scanning electron microscopy details on the structure of the area rugosa and number of caudal papillae in males.

The mitochondrial genome of Dipetalonema gracile from a squirrel monkey in China.

Dipetalonema gracile is a common parasite in squirrel monkeys (Saimiri sciureus), which can cause malnutrition and progressive wasting of the host, and lead to death in the case of massive infection. This study aimed to identify a suspected D. gracile worm from a dead squirrel monkey by means of molecular biology, and to amplify its complete mitochondrial genome by polymerase chain reaction (PCR) and sequence analysis. The results identified the worm as D. gracile, and the full length of its complete mitochondrial genome was 13,584 bp, which contained 22 tRNA genes, 12 protein-coding genes, two rRNA genes, one AT-rich region and one small non-coding region. The nucleotide composition included A (16.89%), G (20.19%), T (56.22%) and C (6.70%), among which A + T = 73.11%. The 12 protein-coding genes used TTG and ATT as start codons, and TAG and TAA as stop codons. Among the 22 tRNA genes, only trnS1AGN and trnS2UCN exhibited the TΨC-loop structure, while the other 20 tRNAs showed the TV-loop structure. The rrnL (986 bp) and rrnS (685 bp) genes were single-stranded and conserved in secondary structure. This study has enriched the mitochondrial gene database of Dipetalonema and laid a scientific basis for further study on classification, and genetic and evolutionary relationships of Dipetalonema nematodes.

Molecular identification and phylogenetic analysis of Dipetalonema evansi (LEWIS, 1882) in camels (Camelus dromedarius) of Iran.

Despite the economic importance of camels, the parasites that affect them have not received adequate attention so far and molecular studies are scarce compared to other livestock. In this study, we characterized peripheral blood microfilariae in 200 healthy one-humped camels (Camelus dromedarius) from south-east Iran by microscopy and molecular tools to receive a more detailed insight into prevalence and species that affect them. Moreover, adult specimens of the filarial nematode Dipetalonema evansi were collected from the carcass of an infected animal. Microscopic examination was performed on Giemsa-stained blood smears, and blood was also spotted on Whatman FTA(®) cards for DNA analysis. Genomic DNA was extracted, and PCR was carried out for the detection of filaroid helminths, followed by sequence analysis of positive samples. Four samples were positive for microfilariae by microscopy, while 16 animals (8 %) were positive by PCR. Sequence analysis revealed D. evansi in all cases. Phylogenetic analysis of a cytochrome C oxidase subunit I (COI) sequence of filaroid nematodes showed that most species in a single genus cluster in the same clade; however, D. evansi and D. gracile are not monophyletic and branch rather at the base of the tree. Further studies on the life cycle of D. evansi, specifically the identification of intermediate host(s), have become feasible with the provision of the first specific COI sequences in this study.

Microscopic and molecular detection of Deraiophoronema evansi (Lewis, 1882) in domestic Bactrian camels (Camelus bactrianus) of Mongolia.

Cameline filarosis is an important parasitic disease having an economic impact on the camel industry around the world. However, there has been no study on filarosis in Bactrian camels of Mongolia. Therefore, the aim of the present study was to detect and identify microfilariae of Deraiophoronema evansi (D. evansi) in Bactrian camels from three provinces, located in southern and southwestern Mongolia. Blood samples were obtained from 400 healthy two-humped camels of different ages and both sexes. All blood samples were analysed using a variety of diagnostic techniques. Microfilariae were detected in 30 Bactrian camels (7.5%) by the Knott technique, while 13 Bactrian camels (3.3%) tested positive in a direct smear test. D. evansi was detected in 18 Bactrian camels (4.5%) by PCR assay. Prevalence was shown to be high among Bactrian camels in the age group up to 5 years, while the lowest positive results were obtained for Bactrian camels in the 5-10-year age group and the over 10-year age group. To confirm the morphological identification, D. evansi-COI gene sequences were subjected to phylogenetic analyses. The D. evansi-COI gene sequences from Mongolian two-humped camels were identical to sequences from Iranian one-humped camels and were clustered together with these sequences in the phylogeny. This is the first report of molecular detection and identification of microfilariae of D. evansi in Bactrian camels of Mongolia.

A new species of Dipetalonema (Filarioidea: Onchocercidae) from Ateles chamek from the Beni of Bolivia.

We describe a new species of Dipetalonema occurring in the body cavity of Ateles chamek (Humboldt, 1812) from north-central Bolivia. Morphologic characters serving to separate Dipetalonema yatesi n. sp. from known forms include a vagina vera with a simple tube and thin walls and a left spicule, which possesses a handle shorter than the lamina (ratio 2.7); the latter displays an anterior membranous alae similar in length to the terminal flagellum, a distal extremity of the left spicule within a simple hook and a membrane, phasmids at the basis of the lappets, and heterogeneous muscles occupying the whole cavity. Dipetalonema yatesi n. sp. can be separated from Dipetalonema robini, Dipetalonema gracile, and Dipetalonema graciliformis, between other characters, in having a simple vagina vera instead of a sinuous one, and from Dipetalonema caudispina and Dipetalonema freitasi in having the lamina of the left spicule divided in a membranous alae and a terminal flagellum.

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.

First record of Acanthocheilonema dracunculoides from domestic dogs in Namibia.

Acanthocheilonema dracunculoides was diagnosed in 2 dogs from Windhoek, Namibia, by acid phosphatase staining of microfilariae. This is the 1st record of A. dracunculoides in Namibia.

Apodemus sylvaticus, a new host for Acanthocheilonema viteae (Nematoda: filarioidea).

Susceptibility of Apodemus sylvaticus and A. agrarius to infection with Acanthocheilonema viteae was compared with that of hamsters and jirds. Microfilaremia in A. sylvaticus was first noted on day 52 post-infection (p.i.) and lasted during the course of the study (up to day 150 p.i.). Maximum microfilaremic levels (female worm basis) of A. sylvaticus [mean +/- S.D. (n) = 690 +/- 1288(6)] were considerably higher than those of hamsters [16 +/- 18(6)] and jirds [51 +/- 25(5)]. Adult worm recovery in A. sylvaticus ranged from 2 to 40% of the number of infective larvae inoculated. Worm development in A. sylvaticus resembled that in hamsters and jirds. In contrast, microfilaremia was not detected in, nor adult worms recovered from A. agrarius throughout the study.

Millardia meltada, a new host for Acanthocheilonema viteae and a simple technique for separation of microfilariae from peripheral blood.

Millardia meltada were infected with Acanthocheilonema viteae and examined for their susceptibility. The morbidity of infected M. meltada was low compared with that of jirds. On day 47 post-infection (p.i.), 13 of 14 M. meltada developed microfilaremia. Male M. meltada then showed gradually increasing microfilaremia with a peak level of 7000 per 30 microliters blood at week 20 p.i., which was much higher than that (3000) of male jirds. In contrast, microfilarial densities of female M. meltada were markedly low with a peak level of 200 during weeks 10-12 p.i. A simple centrifugation technique with Lympholyte-M was devised for microfilarial separation from the peripheral blood of infected M. meltada and yielded approximately 17 x 10(5) viable microfilariae from 1 ml of blood. This method also makes it possible to collect microfilariae from the same individuals repeatedly. M. meltada, coupled with this microfilarial separation technique, serves as a useful animal model for microfilarial studies of A. viteae.

Up-regulation of extracellular copper/zinc superoxide dismutase mRNA after transmission of the filarial parasite Acanthocheilonema viteae in the vertebrate host Meriones unguiculatus.

The gene encoding the cytoplasmic copper/zinc superoxide dismutase (AVSOD1) from the filarial parasite Acanthocheilonema viteae was isolated from a genomic DNA library using a degenerate oligonucleotide probe. Additionally, cDNAs of the AVSOD1 and the secreted extracellular SOD (AVSOD2) were both cloned by RT-PCR, and the AVSOD2 was expressed at high levels in E. coli. The amino acid sequence of the AVSOD1 is 89.5 and 87.5% identical to that of the corresponding enzymes of Brugia pahangi and Onchocerca volvulus, respectively. In contrast, the AVSOD2 shows a lower degree of identity to the other filarial SODs and is extensively glycosylated. RT-PCR studies demonstrate the expression of both SOD subtypes in all developmental stages of A. viteae and indicate up-regulation of the AVSOD2 expression after transmission from the vector to the definitive host. This suggests an enhanced requirement for SOD activity in post-infective larval stages and adults of A. viteae. ELISAs performed with purified recombinant AVSOD2 show that the AVSOD2 is not a major target for the immune system in naturally infected jirds.

Kinetics of Dipetalonema viteae infections established by surgical implantation of adult worms into hamsters.

Native LVG strain hamsters were infected with Dipetalonema viteae by the surgical implantation of adult worms. Groups of hamsters received either 50 male, 50 female, 50 male plus 50 female or 25 male plus 25 female worms per hamster. Approximately 50% of the transferred worms became established in the recipient hosts regardless of the number or sex of the worms implanted. Microfilaremia occurred in recipient hamsters within 1 week after the transfer of female or male plus female worms. This microfilaremia became negative on week 9 post-transfer and no microfilaremia developed in these hamsters following a secondary challenge infection of male plus female worms. Hamsters whose primary infection consisted solely of male worms developed a microfilaremia when challenged with male plus female worms.

Trypanosomes and microfilariae in feral owl and squirrel monkeys maintained in research colonies.

A group of 358 owl and squirrel monkeys imported from Colombia, Peru, and Bolivia for the U.S. Agency for International Development Malaria Vaccine Development Program was examined for trypanosomes and microfilariae. Trypanosoma rangeli, isolated by hemoculture from Aotus nancymai, Saimiri b. boliviensis, and S. b. peruviensis, accounted for 76.6% of all trypanosome infections. Trypanosoma cruzi was isolated from 25 of 194 S. b. boliviensis, including two mixed infections with T. rangeli. Identifications of trypanosomes were confirmed by blinded tests with a panel of five rRNA probes on a subsample of cultures identified morphologically. Although no trypanosomes were isolated from Aotus vociferans or A. lemurinus griseimembra, positive serologic responses to T. cruzi were observed by indirect immunofluorescence assay in all species of monkeys examined and ranged from 42.1% among S. b. peruviensis to 92.3% among A. vociferans. Among T. rangeli-infected monkeys, 43.7% were seronegative for T. cruzi. No microfilariae were found in S. b. boliviensis or A. l. griseimembra. Mansonella barbascalensis and Dipetalonema caudispina were observed in A. vociferans, M. panamensis in A. nancymai, and M. saimiri and D. caudispina in S. b. peruviensis. Such naturally occurring infections in imported animal models are potential sources of accidental transmission to animal handlers and uninfected laboratory animals and can introduce confounding variables into otherwise well-planned and well-executed studies.

Canine filarial infections in north Taiwan.

To assess the current status of Dirofilaria immitis infection and to determine whether there were other canine filarial infections in north Taiwan, postmortem examination was conducted in 180 stray dogs more than 12 months old. Blood and serum samples were examined using a modified Knott's test and an antigen-detecting enzyme-linked immunosorbent assay (ELISA) kit, respectively. Filarial infection was found in 60.6% of the dogs: 55% with D. immitis and 12.2% Dipetalonema reconditum. Moreover, the ELISA was determined to be more sensitive than the Knott's test. Although canine heartworm infection in Taiwan has been attributed to the unrestricted import of dogs from endemic areas, the results of this study indicate that transmission of D. immitis and Dip. reconditum may occur indigenously in the local canine population. This study is also the first record of Dip. reconditum in Taiwan.

Experimental transmission of Dipetalonema dracunculoides (Cobbold 1870) by Rhipicephalus sanguineus (Latreille 1806).

A dog naturally infected with Dipetalonema dracunculoides and having a microfilaremia of 6050 microfilariae per mm3 of blood was used as source of infection. Experimentally cultivated nymphs of Rhipicephalus sanguineus were fed on the donor dog. Once engorged, ninety-three nymphs were removed and kept at 30 degrees C and RH 90% until they moulted to the adult stage. To study the development of microfilariae in the vector, ten ticks were dissected at Day 37 post-infection. Adult infected ticks were fed on two uninfected dogs. The observed pre-patent periods were 69 and 76 days. Trans-stadial transmission of Dipetalonema dracunculoides by Rhipicephalus sanguineus was demonstrated.

Exfoliative colpocytology: a method for the diagnosis of Capuchin monkey filariasis?

During vaginal fluid examinations (Papanicolaou) to study the physiological sexual cycle of Cebus sp., abundant Dipetalonema gracile microfilariae (110-160 microns x 4-5 microns, without a sheath) were encountered in the genital fluid, but not in peripheral blood. Considering the great difficulty in diagnosing this obscure parasitosis, exfoliative colpocytology was found to be an efficient diagnostic.

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.

Canine filariasis. Importance and transmission in the Baix Llobregat area, Barcelona (Spain).

Field and laboratory studies were performed in order to assess the degree of canine dirofilariasis caused by Dirofilaria immitis (Leidy) in the Baix Llobregat region, a fluvial area near Barcelona, Spain. A total of 188 dogs were sampled between May and August of 1994. Three main areas were chosen: the Western Delta, the Eastern Delta and the Northern zone. Simultaneously, a mosquito sampling programme was carried out with CO2 light traps, to search for infective larvae (L3) of D. immitis. Of the 188 dogs sampled, 38 were positive for at least one of the three filaria found: D. immitis 12.8%, Dipetalonema reconditum (Grassi) 3.7% and Dipetalonema dracunculoides (Cobbold) 2.7%. Only 1.1% showed a mixed infection of both D. immitis and D. dracunculoides. Although Dirofilaria repens Raillet et Henry has been found in Spain, it was not found in this study. Comparing the three zones of the Baix Llobregat, the Eastern Delta showed the highest level of D. immitis (35.3%), probably due to the presence of Aedes caspius (Pallas). Despite the effort in sampling the mosquito population, D. immitis was not found in any of the 2001 females dissected, belonging to 5 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.

Epidemiological aspects of filariosis in dogs on the coast of Paraná state, Brazil: with emphasis on Dirofilaria immitis.

The present study determined the prevalence and geographical distribution of Dirofilaria immitis and other filariae, from dogs in littoral areas of Paraná state, in Brazil. This survey spanned eight months, between 1998 and 1999, and was also designed to compare the efficacy of different tests for diagnosis of heartworm infection in that area. Blood samples were collected from 256 native-owned dogs distributed along the Paraná coastal area. Five diagnostic procedures were used: direct smear examination, the Knott's modified test, filtration assay, and two heartworm antigen detection kits. A follow-up imaging exam was performed to support the heartworm diagnosis. The imaging diagnosis included radiographic and ultrasonographic exams of six dogs that had positive results for the heartworm antigen detection kits, but showed different microfilarial burdens. The presence and severity of radiographic and ultrasonographic signs were compared with the results obtained in microfilariae detection and antigen tests. Diagnostic parasitology results indicated that 31.25% of the dogs were microfilaremic. Three different microfilariae were recovered: D. immitis, Dipetalonema reconditum, and the third (mf3) was not identified. D. reconditum was the species with the highest prevalence: 22.6%. In general, D. immitis prevalence was 5.47% (28.57% occult infections), but it varied along the coast and the range was from 0 to 20%. No correlation could be established between the overall scores for microfilarial counts (small or large numbers) and the severity of radiographic results or the likelihood of detecting filariae in the pulmonary artery using echocardiography. The finding of a different type of microfilaria (mf) suggested the existence of a third species in Paraná state, whose prevalence was 4.68%. These results show that to obtain a reliable diagnosis of heartworm infection, antigen detection kits are indicated. Knott's test or filtration should be performed to confirm microfilaremia and not for diagnosis of heartworm infection. Imaging tests support parasitology exams and add more about severity of infection. The northern areas, specially Guaraqueçaba and Ilha das Peças, presented the highest number of heartworm-infected dogs.