Molecular and morphological characterization of three new species of avian Onchocercidae (Nematoda) with emphasis on circulating microfilariae.

BACKGROUND: Blood parasites have been the subject of much research, with numerous reports of the presence of microfilariae in the peripheral blood (circulating microfilariae) of birds belonging to many orders. Current limitations in molecular characterization methods and species identification using morphological characters of circulating microfilariae are major obstacles to improving our understanding the biology of Filarioidea species, particularly in wildlife. The aim of this study was to partially fill these gaps, with particular emphasis on morphological features of microfilariae, which are the most readily accessible stages of these pathogens. METHODS: Peripheral blood samples of 206 birds belonging to genera Acrocephalus (five species) and Sylvia (five species) were examined using the buffy coat method to process the blood samples for the presence of microfilariae. Positive birds were dissected to collect adult nematodes. Microfilariae and adult nematodes were described, and sequences of their mitochondrial cytochrome c oxidase subunit I and nuclear 28S rDNA gene fragments were obtained and used for molecular characterization and Bayesian phylogenetic inferences. RESULTS: Overall prevalence of microfilariae was 2.9%. Microfilariae were found in the blood samples from six birds (2 Acrocephalus scirpaceus and 1 each of A. arundinaceus, Sylvia atricapilla, S. borin and S. curruca), which were dissected. All parasite species observed were new. Eufilaria acrocephalusi sp. n. and Eufilaria sylviae sp. n. were present in subcutaneous, peritracheal and periesophageal connective tissues in A. scirpaceus and S. borin, respectively. Splendidofilaria bartletti sp. n. was found in finger joins of S. atricapilla. Illustrations of microfilariae and adult nematodes are shown, and morphological and phylogenetic analyses identified the DNA barcode haplotypes that are associated with these species. Phylogenetic analysis places the parasites of different genera in different closely related clades. CONCLUSIONS: Adult nematode morphological characters, which have been traditionally used in the taxonomy of Filarioidea species, have a phylogenetic value. Importantly, in our study parasites of different genera were readily distinguishable based on the morphology of their microfilariae. The link between molecular and morphology data requires more study in Filarioidea species research, particularly because this approach provides new knowledge on species identity using only readily accessible blood stages (microfilariae), thereby avoiding host dissection and thus minimizing harm to wildlife during research.

Cercopithifilaria spp. in dogs in Sardinia Island (Italy).

A survey on Cercopithifilaria spp. was carried out on owned and kennelled dogs in Sardinia, Italy. A total of 180 dogs were sampled and tested by microscopic detection or PCR of dermal microfilariae in skin snip sediments. The overall prevalence for Cercopithifilaria spp. at both microscopy and molecular tests was 9.4 % (17/180), while 8.3 % (15/180) of dogs scored positive at microscopic detection of sediments only. Of the 225 microfilariae measured, 212 were identified as Cercopithifilaria bainae and the remaining as Cercopithifilaria sp. II. All samples were molecularly processed for specific amplification of cytochrome oxidase subunit 1 (cox1) and ribosomal 12S gene fragments. The Basic Local Alignment Search Tool analysis of the cox1 and 12S sequences here obtained showed a high nucleotide similarity (99 and 100 %, respectively) with those of C. bainae available in GenBank. In particular, cox1 haplotype I (HI; n=14), haplotype HXVIII (n=2), and a new haplotype, named HXIX (n=1), differing for a single polymorphism from HI, were detected. This study reports data on the occurrence, distribution, and genetic makeup of C. bainae and Cercopithifilaria sp. II infesting dogs in Sardinia, suggesting that these filarioids are spread in areas where Rhipicephalus sanguineus sensu lato ticks occur.

Morphometric analyses of canine blood microfilariae isolated by the Knott's test enables Dirofilaria immitis and D. repens species-specific and Acanthocheilonema (syn. Dipetalonema) genus-specific diagnosis.

BACKGROUND: Considering the increasing importance of small animals travel medicine and the spread of filariae with zoonotic potential to non-endemic European areas, routine filarial diagnosis in dogs is becoming important. Dirofilaria immitis, D. repens, Acanthocheilonema dracunculoides and A. reconditum are the most common canine filarial nematodes presenting blood circulating microfilariae (mf) which can be differentiated to species level by the acid phosphatase activity patterns or by PCR. Available data on the size of the mf vary considerably in the literature. The aim of this study was to validate morphometric criteria for filarial identification in blood samples of dogs after concentration of mf with the modified Knott's technique. METHODS: Morphometric analysis of 10 mf from samples identified to species level by acid phosphatase activity and partially confirmed by PCR were performed with specimens from 377 dogs. RESULTS: The mean length and width of D. immitis mf from 60 dogs were 301.77 ± 6.29 µm and 6.30 ± 0.26 µm, of D. repens mf from 171 dogs 369.44 ± 10.76 µm 8.87 ± 0.58 µm, of A. dracunculoides mf from 133 dogs 259.43 ± 6.69 µm and 5.09 ± 0.47 µm and of A. reconditum mf from 13 dogs 264.83 ± 5.47 µm and 4.63 ± 0.52 µm.For a subset of 30 samples, morphometric analysis was repeated with identical results in two laboratories. Furthermore, the size of mf concentrated and fixed by the Knott's technique was shown to be stable over 105 days. CONCLUSIONS: The Knott's test enables to clearly distinguish between D. immitis, D. repens and Acanthocheilonema spp. However, due to the overlapping size ranges of A. dracunculoides and A. reconditum, biochemical or molecular methods are required to distinguish these two species.

Differentiation of the microfilariae of Dirofilaria immitis and Dirofilaria repens in stained blood films.

An examination was made of Giemsa-stained microfilariae in thin blood films from (n = 9) dogs naturally or experimentally infected with Dirofilaria immitis or Dirofilaria repens. Morphological measurements (total length, length of cephalic space, anterior end to nerve ring and last body nucleus, and nucleus-free tail tip) were made on 2-6 microfilariae from each dog with the use of digitally captured images and imaging software. The microfilariae of D. repens were significantly greater (P < 0.001) in all measured dimensions except for the length of the cephalic space, which was significantly shorter (P < 0.001) than that of D. immitis. The cephalic space of D. repens was characterized by being short and routinely being terminated by a distinct pair of nuclei that were separate from the remaining somatic nuclei of the microfilaria. The cephalic space of the smaller microfilaria of D. immitis was longer and did not have the distinct nuclei separated from the somatic column nuclei near the anterior end. The character of the cephalic space seems to be a criterion that could be routinely used for the easy differentiation of these 2 microfilariae in stained blood films.

Ochoterenella esslingeri n. sp. (Nematoda: Onchocercidae: Waltonellinae) from Bokermannohyla luctuosa (Anura: Hylidae) in Minas Gerais, Brazil, with notes on Paraochoterenella Purnomo & Bangs, 1999.

The waltonelline Ochoterenella esslingeri n. sp., a filarial parasite of the anuran Bokermannohyla luctuosa in Minas Gerais, Brazil is described. Several characters distinguish this new species from the 15 species presently included in the genus: the cuticular ornamentation of the female that is restricted to the posterior region of the body, the irregular arrangement of the small, rounded bosses, the postoesophageal vulva, the short glandular oesophagus, the size and shape of the microfilariae, the long left spicule and high spicular ratio. Irregularly arranged, tiny, rounded bosses are common in the monotypic genus Paraochoterenella from an Indonesian ranid, which is not well defined but likely valid. In the Neotropical Realm, the type hosts of the species of Ochoterenella are Hylidae (O. esslingeri n. sp.), Leptodactylidae (two species) and the remaining 13 species were described from the giant toad Rhinella marina (Bufonidae).

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.

Ivermectin disrupts the function of the excretory-secretory apparatus in microfilariae of Brugia malayi.

Ivermectin (IVM) is a broad-spectrum anthelmintic used in filariasis control programs. By binding to nematode glutamate-gated chloride channels (GluCls), IVM disrupts neurotransmission processes regulated by GluCl activity. IVM treatment of filarial infections is characterized by an initial dramatic drop in the levels of circulating microfilariae, followed by long-term suppression of their production, but the drug has little direct effect on microfilariae in culture at pharmacologically relevant concentrations. We localized Brugia malayi GluCl expression solely in a muscle structure that surrounds the microfilarial excretory-secretory (ES) vesicle, which suggests that protein release from the ES vesicle is regulated by GluCl activity. Consistent with this hypothesis, exposure to IVM in vitro decreased the amount of protein released from microfilariae. To better understand the scope of IVM effects on protein release by the parasite, three different expression patterns were identified from immunolocalization assays on a representative group of five microfilarial ES products. Patterns of expression suggest that the ES apparatus is the main source of regulated ES product release from microfilariae, as it is the only compartment that appears to be under neuromuscular control. Our results show that IVM treatment of microfilariae results in a marked reduction of protein release from the ES apparatus. Under in vivo conditions, the rapid microfilarial clearance induced by IVM treatment is proposed to result from suppression of the ability of the parasite to secrete proteins that enable evasion of the host immune system.

An atypical microfilaria in blood samples from inhabitants of Brazilian Amazon.

An unidentified microfilaria sharing characteristics with Mansonella ozzardi and Onchocerca volvulus was detected in blood samples from seven human volunteers, inhabitants of a community in the border of Amazonas and Acre State. They were detected during epidemiological studies carried out in some communities along Antimary, Acre, and Purus Rivers in the Brazilian Amazon. The most striking difference was presented in the shape of the cephalic space from this microfilaria which was different from those of M. ozzardi and with similarities to O. volvulus in this region, but no remarkable differences were observed at the caudal region. More accurate studies are being carried out in order to provide additional data and supporting evidences before establishment of a new species can be done.

Microfilariae in Galápagos penguins (Spheniscus mendiculus) and flightless cormorants (Phalacrocorax harrisi): genetics, morphology, and prevalence.

Galapagos penguins (Spheniscus mendiculus) and flightless cormorants (Phalacrocorax harrisi) live in small, isolated populations on the westernmost islands of Isabela and Fernandina in the Galápagos Islands, Ecuador. Between August 2003 and February 2005, 4 field trips, 2 in the cool, dry season (August 2003 and August 2004) and 2 in the hot, rainy season (March 2004 and February 2005), were undertaken; 298 Galápagos penguins and 380 cormorants were sampled for prevalence and intensity of hemoparasites. Microfilariae were found in both the penguins and the cormorants. Blood smears were negative for the presence of other species of hemoparasites. Overall prevalence of microfilariae across seasons was 42.0% in cormorants and 13.8% in the penguins. Intensity of infection was generally low (mean = 3.2-31.7 in 25 fields across seasons and species) with the exception of a few individuals with markedly high intensities of parasites (>300 in 25 fields in 1 cormorant). Prevalence of microfilariae increased significantly over the 4 sampling periods for cormorants, but not for penguins. Prevalences were significantly higher in cormorants than in penguins for 3 of the 4 collecting trips. Male penguins had higher prevalences than females; however, there were no gender differences in cormorants. No relation was detected between body mass and either presence or intensity of parasitism. Morphological characteristics of the microfilariae are also described and specimens from each host species were similar in all characters measured. DNA sequence data from the mitochondrial cytochrome c oxidase subunit I gene were consistent with the morphological evidence and together demonstrate that the penguins and cormorants are likely to be infected with the same species of microfilariae.

Molecular evidence for host specificity of parasitic nematode microfilariae in some African rainforest birds.

Here we describe, determine the prevalence, and examine the host-specificity of some parasitic nematode microfilariae in selected bird species from West and Central Africa. We used microscopy to determine the prevalence of microfilariae in 969 host individuals representing 121 rainforest bird species from Cameroon, Côte d'Ivoire and Equatorial Guinea. Thirteen (11%) of these potential host species harboured microfilariae, and 35 individuals (3.6%) were infected. From the 35 infected individuals, we identified eight distinct morphological microfilarial forms. Sixteen of the 35 infected individuals were of one host species, the Fire-crested Alethe (Alethe diademata), at a prevalence rate of 62%. To examine host and geographical specificity, we sequenced a portion of the LSU rDNA gene from representative microfilariae drawn from different hosts and collecting locations. Identical sequences of the nematode LSU rDNA gene were found in A. diademata collected from locations in Côte d'Ivoire and Equatorial Guinea, locations separated by the Dahomey Gap and associated with different hypothesized refugial areas. In contrast, several other bird species collected at the same sites harboured different microfilaria lineages. We sequenced the mitochondrial ATP synthase genes of the host species A. diademata, and found a 5.4% sequence divergence between the birds sampled in Côte d'Ivoire, and those from Cameroon. Thus, despite this split between the two populations, they harbour microfilariae with identical lineages. These data provide evidence that the microfilariae found in A. diademata may be highly host specific. This apparent specificity may have important implications for the evolutionary and ecological interactions between parasitic nematodes and their avian hosts.

Description of adult and fourth-stage larva of Litomosoides navonae n. sp. (Nematoda: Onchocercidae), a parasite of five species of sigmodontine rodents from northeastern Argentina.

This study describes a new species of Litomosoides Chandler, 1931, parasitic in five different sigmodontine rodents from Misiones, Chaco and Formosa provinces of Argentina. The fourth-stage (L4) larva (male and female) is also described. L. navonae n. sp. exhibits: a bottle-shaped buccal cavity; a buccal capsule with irregularly crenate external walls; four externo-labial papillae and one ventral cephalic papilla; a well differentiated oesophagus; and sigmodontis-type spicules. The microfilaria is fusiform, with a large sheath. The L4 has a buccal capsule which is relatively longer than that of the adults, with narrower walls and a bottle-shaped lumen. It was observed in this species that the oesophagus/body-length ratio increases from larva to adult (female ratio 26.2-28.3 in larva; 88.4 in adults), and the vulva appears to move further posterior to the oesophago-intestinal junction (200-300 microm in larvae vs a mean of 600 microm in adults). L. navonae was found parasitising: Nectomys squamipes from the Reserve UNLP Valle del Arroyo Cuñá Pirú, Misiones; Oligoryzomys chacoensis, Holochilus chacarius and Akodon azarae bibianae from the marshes of Arroyo Bellaco, El Colorado, Formosa; and O. fornesi and H. chacarius from Selvas del Río de Oro (Chaco). Both N. squamipes and H. chacarius harbour other filarioids species, i.e. L. kohnae Bain, Petit & Diagne, 1989 and L. patersoni (Mazza, 1928), respectively, throughout their range, but these filarioids are readily differentiated from L. navonae. These well-differentiated filarial species found in Nectomys and Holochilus could indicate how isolated the populations of rodents are and could be interpreted either as: (a) an early point in the speciation processes that could be taking place in these hosts; or (b) extra support for the capture phenomenon theory of the evolution of Litomosoides. New regions, such as southern Brazil and northern Argentina, need to be studied in order to clarify these alternatives.

Hsp90 is essential in the filarial nematode Brugia pahangi.

The development of a compound with activity against filarial nematodes (a 'macrofilaricide') has been a long-standing goal of the World Health Organization. However, adult filariae have proved remarkably difficult to kill. To some extent this reflects a lack of understanding of key pathways and processes in filarial nematodes that may be suitable targets for chemotherapy. In this paper we show that geldanamycin (GA), a specific inhibitor of the activity of the heat shock protein 90 (Hsp90) family, kills adult worms and microfilariae (Mf) of Brugia pahangi at nanomolar concentrations. In addition, release of Mf from adult worms is inhibited within 24 h of exposure to GA and is not recoverable, demonstrating that GA effectively sterilises the worm. Similar results were obtained with a second filarial worm Acanthocheilonema viteae. In contrast GA has no effect on the free-living nematode Caenorhabditis elegans despite a high degree of conservation between the nematode Hsp90 sequences. In keeping with these findings, Brugia Hsp90 binds GA in a solid phase pull-down assay while the binding of C. elegans Hsp90 to immobilised GA is undetectable. In other eukaryotes, GA is known to bind in the N-terminal ATP pocket of Hsp90, disrupting its interactions with client proteins which are then targeted for degradation via the proteasome pathway. Thus, Hsp90 or some of its client proteins may provide novel targets for the chemotherapy of filarial infection.

The morphological discrimination of microfilariae of Onchocerca volvulus from Mansonella ozzardi.

There is no published account which allows the morphological discrimination of microfilariae of Onchocerca volvulus and M. ozzardi from each other. However, they occur together in parts of Brazil and Venezuela, and presumably there is always the possibility that migration could establish new sympatric populations in the future. The objective of this study was to evaluate simple morphological characters that might be used for species-diagnosis of microfilariae. The conclusions were that the location of microfilariae in the blood or skin, the body size and the nucleation of the nerve ring are expected to be useful first indications of species identity, but cannot be used for confident diagnosis. The structure of the cephalic armature (stained with alcian blue) seems to be species specific, but is of limited application because it is often difficult to see. However, the pattern of nucleation of the tail (as expressed by the ratio of the length of the terminal nucleus compared with the length of the tail space) is distinctive and is expected to be diagnostic.

Cytomorphology of filariasis revisited. Expansion of the morphologic spectrum and coexistence with other lesions.

OBJECTIVE: To review the cytomorphologic spectrum of the filarial worm and associated tissue response in 33 cases. STUDY DESIGN: Retrospective analysis was carried out in clinically unsuspected cases of filariasis diagnosed on cytology over a period of 10 years. Twenty-nine aspirate smears from 28 patients were air dried and stained with May-Grünwald-Giemsa stain. Four routine cervical smears and one centrifuged smear of urine were stained with Papanicolaou stain. RESULTS: Microfilariae alone and along with adult gravid females were present in 25 and 4 cases, respectively. In one case both adult male and female worms with microfilariae and eggs were seen. The diagnosis was based on the presence of eggs alone in one case and fragments of female worms in two. Four of these cases were neoplastic lesions, and microfilariae were found incidentally. In one case of splenomegaly microfilariae were seen along with Leishman-Donovan bodies. CONCLUSION: Filariasis can be diagnosed on cytology by demonstrating microfilariae, a male or female worm, or eggs alone. It can be seen in association with neoplastic lesions and rarely with other parasitic infections.

Variation in microfilariae and infective stages of two types of Wuchereria bancrofti from the Thai-Myanmar border.

Comparative morphometric and morphological studies of microfilariae and infective stages were undertaken in nocturnally periodic and subperiodic Wuchereria bancrofti. For microfilariae, the body dimensions of nocturnally periodic (NP) were significantly smaller than nocturnally subperiodic (NSP), i.e., body length 268.03+/-14.75 microm (NP), 307.61+/-11.52 microm (NSP); cephalic space length 4.21+/-0.62 microm (NP), 5.32+/-0.79 microm (NSP); head to nerve ring 49.39+/-5.43 microm (NP), 57.40+/-4.46 microm (NSP); innenkörper length 33.05+/-5.89 microm (NP), 44.02+/-8.71 microm (NSP); cephalic space width 4.28+/-0.59 microm (NP), 6.04+/-0.68 microm (NSP); body width at nerve ring 5.01+/-0.57 microm (NP), 7.45+/-0.75 microm (NSP). The number of nuclei between the cephalic space and nerve ring of NP (66.67+/-5.19) was also significantly less than in NSP (94.74+/-6.95). For infective stages, the body dimensions of NP were significantly smaller than NSP, i.e., body length 1632.50+/-131.48 microm (NP), 2002.63+/-222.60 microm (NSP); head to nerve ring 103.09+/-7.47 microm (NP), 122.44+/-9.62 microm (NSP); head to oesophago-intestinal junction 567.69+/-94.84 microm (NP), 666.75+/-110.08 microm (NSP); body width at oesophago-intestinal junction 23.15+/-1.55 microm (NP), 26.78+/-1.62 microm (NSP). It is too early to infer the NP type as an additional sibling species of W. bancrofti but it is reasonable to treat it as a new variety and additional work is needed to clarify its status.

Cercopithifilaria shohoi n. sp. (Nematoda: Filarioidea) from the relict Bovidae, Capricornis crispus, in Japan.

Cercopithifilaria shohoi n. sp. was found in the relict bovid, Capricornis crispus, in Japan, and is described and compared with other species in the genus. Adult male and female worms were found in subcutaneous tissues of the trunks of 6 serows shot in Mt. Zao, Yamagata Prefecture, in the northern part of Honshu, the largest island of Japan. The one complete male found was 19.7 mm long, and the five females were 31.6-50.9 mm long. Unsheathed or sheathed microfilariae 104-122 microns long were taken from the females. One microfilaria was found in the sediment of the preservation solution of the tissues, but none were found in the blood of the infected serows, so microfilariae may be limited to the skin. Males of this species had one pair of papillae between perianal and subterminal groups of caudal papillae. In having this intermediate pair, C. shohoi n. sp. resembled species such as C. faini from an African bovid and C. rugosicauda from a European deer. From its morphological characteristics, C. shohoi n. sp. seems to be one of the more primitive species in the genus Cercopithifilaria.

Surface area calculation for Onchocerca volvulus microfilariae.

The pathogenesis of tissue damage in filarial infections involves the generation of inflammatory mediators by interaction of parasite surfaces with the host immune response. In the presence of a heavy parasite burden, the tremendous potential for development of adverse local or systemic inflammatory responses may be appreciated by knowledge of the cumulative surface area represented by circulating microfilariae. The approximate surface area of a single microfilariae of Onchocerca volvulus was calculated using mathematical formulae for standard geometrical shapes and values derived from serial measurements of electron micrographs. Numerical values for the surface area represented by large numbers of microfilariae are given and compared to surface areas of the host which can harbor many millions of microfilariae.