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.

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.

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.

Dipetalonema evansi infection in camels of Iran's central area.

Totally 294 dromedary camels of different ages and both sexes slaughtered at slaughterhouses in Yazd, Isfahan and Kerman provinces were inspected for infection with Dipetalonema evansi. Blood smears of all camels and carcasses of 125 of them (100 from Isfahan and 25 from Yazd) were studied for larva and adult forms of the parasite. Microfilariae were found in peripheral blood smears of 38 out of 294 (12.92%) tested camels, while 20 out of 125 camels (13.89%) harbored D. evansi adult worms in at least one region in their testicle, epididymis, spermatic cord, lung and heart. Two of infected males had adult forms of the parasite in all studied organs simultaneously. Pathological study of infected tissues revealed sections of parasite, severe acute and chronic inflammation, fibrosis and atrophy. D. evansi is endemic and constitutes an important health problem to camels in Iran's central desert, resulting in impaired working capacity and lowered productivity.

Hippobosca longipennis--a potential intermediate host of a species of Acanthocheilonema in dogs in northern India.

BACKGROUND: Hippobosca longipennis (the 'dog louse fly') is a blood sucking ectoparasite found on wild carnivores such as cheetahs and lions and domesticated and feral dogs in Africa, the Middle East and Asia, including China. Known as an intermediate host for Acanthocheilonema dracunculoides and a transport host for Cheyletiella yasguri, it has also been suggested that H. longipennis may be a vector for other pathogens, including Acanthocheilonema sp.? nov., which was recently reported to infect up to 48% of dogs in northern India where this species of fly is known to commonly infest dogs. To test this hypothesis, hippoboscid flies feeding on dogs in Ladakh in northern India were collected and subjected to microscopic dissection. RESULTS: A total of 12 infective larvae were found in 10 out of 65 flies dissected; 9 from the head, 2 from the thorax and 1 from the abdomen. The larvae averaged 2, 900 (± 60) µm in length and 34 (± 5) µm in width and possessed morphological features characteristic of the family Onchocercidae. Genetic analysis and comparison of the 18S, ITS-2, 12S and cox-1 genes confirmed the identity of the larvae as the Acanthocheilonema sp.? nov. reported in dogs in Ladakh. CONCLUSION: This study provides evidence for a potential intermediate host-parasite relationship between H. longipennis and the canine Acanthocheilonema sp.? nov. in northern India.

First report of Acanthocheilonema spirocauda in the Mediterranean monk seal (Monachus monachus).

The Mediterranean monk seal (Monachus monachus) is one of the world's most endangered marine mammals. The largest population is located mainly throughout the Aegean and Ionian islands and along the coastline of southern continental Greece. We report the findings of a necropsy and discuss their potential importance to the conservation of the species. The adult female monk seal appeared to be in a good nutritional state. The main necropsy findings were injuries consistent with a violent and sudden death, including three round wounds on the ventral surface of the body and several hematomas, as well as a decomposing male fetus in the uterus. Two nematodes were found in the right ventricle of the heart; no abnormalities were observed in the lungs, pulmonary vessels, or heart. The nematodes were identified as Acanthocheilonema spirocauda. This is the first report of infection of the Mediterranean monk seal with A. spirocauda, even though this is the most common heartworm found in most pinnipeds worldwide. This parasite should be considered in health care monitoring projects of this endangered species.

Characterization of the heartworm Acanthocheilonema spirocauda (Leidy, 1858) Anderson, 1992 (Nematoda: Onchocercidae) in Scandinavia.

The heartworm Acanthocheilonema spirocauda (Leidy, Proc Acad Nat Sci Philadelphia 10:110-112, 1858) Anderson, 1992 is described from material collected from harbour seals in Scandinavia and compared with types and other specimens described by Anderson (Can J Zool 37:481-493, 1959) from harbour seals in eastern USA. Most morphometric characters of the material from USA fall within the ranges established for the Scandinavian one. Some intraspecific variability in the organisation of papillae on the male tail was detected among the Scandinavian specimens. Differences between the specimens from Scandinavia and Eastern USA are also found in the organisation of papillae on the tail of males and females. An excretory pore was not discernible, but a clearly hemizonid-like structure is described. For the first time, scanning electron micrographs present external morphological structures of the species.

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.

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.

Specific polymerase chain reaction for differential diagnosis of Dirofilaria immitis and Dipetalonema reconditum using primers derived from internal transcribed spacer region 2 (ITS2).

Both Dirofilaria immiti and Dipetalonema reconditum may be found in blood of infected dogs but it is not easy to distinguish D. immitis from D. reconditum in morphology. We cloned and sequenced the contiguous internal transcribed spacer (ITS) region, ITS1-5.8S-ITS2, of these two different parasites and published on GenBank as AF217800 for D. immiti and AF217801 for D. reconditum in this study. We designed two pairs of specific primers derived from ITS2 being used for polymerase chain reaction (PCR). The amplicons of ITS2 from D. immiti and D. reconditum are 302 and 348bp, respectively. Moreover, the limitation for amplifying ITS2 gene using this PCR demonstrated that 1 x 10(-2) microfilaria of each species of parasite smashed or even with mixed samples could be detected and the PCR products were predicted as the same as that described above. Thus, D. immiti and D. reconditum could be differentially diagnosed by this specific PCR. Seventeen clinical cases were evaluated and all of them were correctly identified. In this study, ITS1-5.8S-ITS2 of D. immiti or D. reconditum were the first time sequenced and analyzed. No significant similarity of ITS1 and ITS2 between D. immiti and D. reconditum could be observed.

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.

Distribution, prevalence, and relative risk of filariasis in dogs from the State of Washington (1997-1999).

Using antigen capture and filter tests, 6,078 dogs throughout the state of Washington were examined for filariasis between July 1, 1997 and October 31, 1999. In western Washington, 791 males and 901 females examined were outdoors, not on prophylaxis, and had traveled out of the state; 6/791 (0.8%) males and 7/901 (0.8%) females were infected with Dirofilaria immitis (D. immitis), and one (0.1%) male and one (0.1%) female were infected with Dipetalonema reconditum (D. reconditum). There were also 392 males and 362 females examined that were outdoors, not on prophylaxis, and had not traveled out of western Washington. One (0.1%) female was infected with D. immitis, and two (0.5%) males and one (0.2%) female were infected with D. reconditum. In eastern Washington, 707 males and 826 females examined were outdoors, not on prophylaxis, and had traveled out of the state; 9/707 (1.0%) males and 4/826 (0.5%) females were infected with D. immitis, and no D. reconditum was found. There were also 376 males and 412 females examined that were outdoors, not on prophylaxis, and had not traveled out of the state. Three (0.8%) males and three (0.7%) females had D. immitis. One (0.2%) female had D. reconditum. Distribution of D. immitis-infected, nontravel dogs in eastern Washington was only found between 120 degrees and 119 degrees west longitude in the communities of Richland, Moses Lake, Okanogan, and Omak. Enzootic transmission of D. immitis and D. reconditum is occurring in both eastern and western Washington.

[Prevalence of canine filariasis by Dirofilaria immitis and Dipetalonema reconditum in Maceió, Alagoas State, Brazil]. / Prevalência da filariose canina causada por Dirofilaria immitis e Dipetalonema reconditum em Maceió, Alagoas, Brasil.

A survey on the prevalence of Dirofilaria immitis and Dipetalonema reconditum was conducted in 1,519 dogs from Maceió and two coastal areas in the State of Alagoas, Northeast Brazil, from 1995 to 1999, by testing for microfilariae in blood. All blood samples were from exclusively domiciled dogs with a known history, showing that the infections were autochthonous, confirming transmission of canine filariasis in these areas. In Greater Metropolitan Maceió, 15 (1.3%) microfilaremic dogs were detected with D. immitis and 15 (1,3%) with D. reconditum. In the southern coastal area there was an estimated prevalence of 12.7% for D. immitis. D. immitis and D. reconditum microfilaria were 298.1 micrometer and 249.2 micrometer long and 7.3 micrometer and 4.4 micrometer wide, respectively. A Witness immunotest that detects D. immitis antigen was used to confirm parasitological results and reveal occult dirofilariasis cases. Of the total 6,579 females examined, 8 (0.1%) Culex quinquefasciatus were observed to be naturally infected with D. immitis larvae. These results proved dirofilariasis transmission in Maceió and demonstrated D. reconditum in the same geographic area.