Morphological and Molecular Data Reveal Two New Species of Viannaia (Nematoda: Viannaiidae), Parasitizing Opossums (Mammalia: Didelphidae) in Mexico.

Two new species of Viannaia from the intestine of the North American opossums, Didelphis virginiana (Virginia opossum), and Philander opossum (gray four-eyed opossum), are described based on morphological and molecular data, through an integrative taxonomic approach. Maximum likelihood and Bayesian inference analyses for each dataset and the concatenated dataset were performed using a mitochondrial cytochrome c oxidase subunit 1 (COI) gene, and the nuclear ribosomal internal transcribed spacer region (ITS1-5.8S-ITS2). The phylogenetic analyses revealed 2 new species that occur in Mexico, one from the western state of Colima and another from the southern state of Chiapas. Our phylogenetic trees for both molecular markers and concatenated datasets yielded similar topologies with high bootstrap values and posterior probabilities. Viannaia is recovered as a monophyletic group, but the family Viannaiidae appears as non-monophyletic, due to the position of Travassostrongylus scheibelorum, similar to previous studies. Finally, the morphology of Viannaia and Hoineffia is discussed.

In Vitro, in Vivo, and Interaction Studies of Nematophagous Fungus Arthrobotrys thaumasia ( Monacrosporium thaumasium) with the Larvae of Trichostrongylides of Sheep.

It is important to isolate potential candidates from the local isolates of nematophagous fungi and to investigate interaction between the fungal strains and gastrointestinal nematodes for the biological control of parasitic nematodes in livestock. In the present study, we assessed the in vitro predatory activity and the viability of isolates of Arthrobotrys thaumasia ( Monacrosporium thaumasium) after passage through the gastrointestinal tract of sheep. The predatory process of a representative isolate selected against the larvae of trichostrongylids was prepared with a scanning electron microscope (SEM). In vitro experiments tested the ability of 9 native isolates of A. thaumasia to prey on larvae of feces of sheep infected with natural mixed nematodes ( Haemonchus contortus, Trichostongylus colubriformis, Marshallagia mongolica). These isolates of A. thaumasia decreased infectivity of third stage infective larvae (L3) by 75.54-99.97%; 7 isolates decreased infectivity by more than 90%. In vivo experiments also demonstrated significant reductions of L3 numbers in the feces treated with the 9 isolates after passing through the gastrointestinal tract of sheep, and these decreases ranged from 51.68 to 88.16%. The isolates tested were re-isolated in 5-g sub-samples of feces from sheep in each treatment group, indicating that these isolates had the capacity to prey upon larvae of trichostrongylids after the passage through gastrointestinal tract. SEM shows that at 6 hr after the larvae were added, including the second stage larvae (L2) and L3 of trichostrongylids, the isolate NBS 005 caught them; at 8 hr after being caught L2 was penetrated by the fungus while penetration of L3 occurred at 12 hr; at 78 hr post-capture L2 was completely destroyed by the fungus while complete digestion of L3 occurred at 84 hr.

Anthelmintic effect of 2H-chromen-2-one isolated from Gliricidia sepium against Cooperia punctata.

Gliricidia sepium is a tropical legume with known anthelmintic-like properties. The aim of this study was to: (1) perform a bio-guided fractionation of an acetonic extract of G. sepium leaves using the egg hatch assay (EHA); (2) elucidate the anthelmintic (AH)-like phytochemical using nuclear magnetic resonance (NMR); and (3) assess the ultrastructural damage of the Cooperia punctata treated eggs. The anthelmintic activity of G. sepium was traced from an acetonic extract using the EHA. Phytochemicals were isolated through silica gel columns and elucidated through spectroscopic measurements (1H and 13C). Final fraction was evaluated with EHA at decreasing concentrations of: 1.100; 0.500, 0.250, 0.125, 0.060, 0.001 and 0.00001 mg mL-1. Egg hatching inhibition was calculated using the formula: 100*(1-HT/HC). The maximal half of effective concentration (EC50) was calculated with GraphPad. Bio-guided isolation procedures lead to the elucidation of 2H-chromen-2-one, which inhibited both hatching and embryo development of C. punctata (EC50 of 0.024 ± 0.082 mg mL-1) (P < 0.05). Scanning and Transmission Electron Microscopy (SEM and TEM) revealed electrodensity alterations and fractures in the eggshell layers. After toxicity evaluations and in vivo assessment, 2H-chromen-2-one can be suggested as a novel AH-phytochemical for reducing larval density in pastures and worm burdens inside the host.

Reappearance of Mecistocirrus digitatus in cattle from the Mexican tropics: prevalence, molecular, and scanning electron microscopy identification.

Mecistocirrus digitatus is a hematophagous abomasal nematode which causes significant blood and production losses in cattle. The objectives of the present study were to: (1) report the reappearance of M. digitatus in cattle from the Mexican tropics using microscopy, scanning electron microscopy (SEM), and molecular identification, and (2) determine the prevalence of M. digitatus in slaughtered adult cattle from the Mexican tropics. Gastroinestinal nematodes (GIN) were recovered from the abomasum of an 8-yr-old cow (Holstein × Zebu) previously diagnosed with Johne's disease. Of 1,254 GIN, 98.8% were identified as M. digitatus and 1.2% as Haemonchus sp. SEM was used to identify ultrastructure features of M. digitatus (oral cavity, cervical papillae, bursa, bursa lobes papillae, male spicules, anus, and female vulva). A conventional PCR method was used to corroborate the morphological findings. The prevalence of adult cattle infected with M. digitatus and Haemonchus sp., determined from 68 adult cattle from different grazing tropical herds, was 38.2% and 8.8%, respectively. Ninety-eight percent of animals infected with M. digitatus presented lesions in their abomasum such as mucosal inflammation, hemorrhage, and ulcers; some of them had necrosis. The current reappearance of M. digitatus in a Mexican herd suggests the possibility of an underestimated prevalence of this nematode amongst grazing cattle.

A simple and efficient method to observe internal structures of helminths by scanning electron microscopy.

Morphological studies of helminths by scanning electron microscopy are generally limited to the external topography of the organisms. In this work, we present a simple technique using ethanol as a cryoprotectant without postfixation in osmium tetroxide that allows for observation of the inner organization of helminths and preserves cellular structures. We tested the technique in three helminths: Echinostoma paraensei, Cruzia tentaculata, and Hassalstrongylus epsilon. The results show that this technique could potentially be applied to study the morphology, ultrastructure, and taxonomy of helminths.

The comparative morphology of Marshallagia marshalli and Ostertagia occidentalis (Nematoda: Strongylida, Trichostrongylidae) by scanning electron microscopy.

The systematics of the Ostertagiinae is unsettled with no agreement on how many genera and species are present in cattle and sheep. Ten species of Ostertagiinae are commonly parasitic in cattle and sheep. In the global fauna, six of 13 ostertagiine genera are endemic to Iran. The life cycle of Ostertaginae is direct and ingested third-stage larvae after exsheatment in the rumen, penetrate the gastric glands in the abomasal mucosa where two parasitic moults occur before the L5 emerges from the gland. In the present work, Marshallagia marshalli and Ostertagia occidentalis, collected from the abomasums of sheep from Mashhad, Iran, is described. The association of light and scanning electron microscopy (SEM) allowed a detailed analysis of the morphology and ultrastructure of these nematodes. The male body length of M. marshalli and O. occidentalis were 9.3-10.20 and 9.60-10.50 mm, respectively. The female body length of M. marshalli and O. occidentalis were 10.10-15.30 and 10.4-15.70 mm, respectively. One of cervical papillae is seen 333 and 250 µm from the anterior end of male and female body surface in O. occidentalis and 287.5 and 200 µm from the anterior end of male and female body surface in M. marshalli, respectively. The size of cervical papillae is 13.3 µm in male and 10 µm in female in O. occidentalis and 9.33 µm in male and 8.57 µm in female in M. marshalli. Some other taxonomic features of M. marshalli and O. occidentalis, such as details of cephalic region, the system of longitudinal and surface cuticular ridges (synlophe), the orientation of rays of the copulatory bursa, localization of vulva, morphology of vulvar flap, and posterior end of females are also documented by SEM.

Morphological diagnosis of infective larvae of Libyostrongylus douglassii (Cobbold, 1882) Lane, 1923 and L. dentatus Hoberg, Lloyd and Omar, 1995 (Nematoda: Trichostrongylidae) of ostriches.

The differentiation of the species of the Libyostrongylus genus is only possible with the obtainment of the adult parasites in the ostriches proventriculus and gizzard. The present work confirms that it is possible to differentiate the infective larvae of L. douglassii and L. dentatus allowing the differential diagnosis of these species by fecal culture. To show this, adult females from both species were collected from ten proventriculus from adult ostriches and separated by species. Both groups were macerated individually added to sterilized feces for standard fecal cultures. The infective larvae were recovered, identified, quantified and measured. All proventriculus analyzed were parasitized by Libyostrongylus spp. and a clear heterogeneous location for each species was observed. The infective larvae from the fecal cultures of macerated L. douglassii presented a mean total length of 874.3+/-33.80 microm, and a short sheath tail (29.5+/-4.11 microm) with acute termination. The infective larvae from the macerated L. dentatus presented mean total length of 856.0+/-43.63 microm, long sheath tail (61.2+/-9.52 microm) with filamentous termination. The mean measures of the tails of both species had a significant difference. The differentiation of the infective larvae of L. douglassii and L. dentatus by fecal cultures will facilitate the diagnosis of both species for further understanding the Libyostrongylus biology.

An exploration of diversity among the Ostertagiinae (Nematoda: Trichostrongyloidea) in ungulates from Sub-Saharan Africa with a proposal for a new genus.

Abomasal nematodes (Ostertagiine: Trichostrongyloidea) representing a previously unrecognized genus and species are reported in African buffalo (Syncerus caffer caffer) from Kenya, Uganda, and South Africa. Africanastrongylus buceros gen. nov. et sp. nov. is characterized by a symmetrical tapering synlophe in the cervical region and a maximum of 60 ridges in males and females. Bursal structure is 2-2-1, with subequal Rays 4/5, massive Rays 8, and Rays 9/10, and a massive dorsal lobe that is reduced in length, laterally and dorsally inflated, and positioned ventral to externodorsal rays. Spicules are tripartite, and the gubernaculum is broadly alate in the anterior. A proconus is present. Among ostertagiines with a 2-2-1 bursa (Cervicaprastrongylus, Hyostrongylus, Mazamastrongylus, Sarwaria, Spiculopteragia, and Teladorsagia) specimens of Africanastrongylus are differentiated from respective genera based on the structure of the cervical synlophe, patterns of dorsal, externodorsal, lateral, and ventral rays, and configuration of the genital cone, gubernaculum, and spicules. Among 13 genera of the Ostertagiinae in the global fauna, 3 are entirely limited in distribution to Africa, including Africanastrongylus, Longistrongylus, and Pseudomarshallagia. Another 5 genera including Cervicaprastrongylus, Hyostrongylus, Marshallagia, Ostertagia, and Teladorsagia are represented as mosaics, with diversity centered in Eurasia or the Holarctic. Genera not represented in the African fauna include Camelostrongylus among Caprinae and some Antelopinae from Eurasia, Mazamastrongylus and Spiculopteragia in Cervidae from the Holarctic and Eurasia, respectively, Orloffia in Cervidae and Bovidae from the Holarctic, and Sarwaria among Tragulidae and Bovinae in southern Asia. The diverse nature of the ostertagiine fauna, with a disproportionate number of endemic genera relative to other regions of the northern hemisphere, may reflect the timing of episodic expansion events for artiodactyls into Africa from Eurasia during the Tertiary and Quaternary.

Mixed infection by Libyostrongylus douglassii and L. dentatus (Nematoda: Trichostrongylidae) in Struthio camelus (Ratites: Struthioniformes) from Brazil with further morphological characterization of adults.

The genus Libyostrongylus includes three species, L. douglassii, L. dentatus and L. magnus that occur as parasites in the proventriculus of Struthio camelus. We confirmed a mixed infection by L. douglassii and L. dentatus in farmed ostriches from the southeast of Brazil for the first time, and provided new information on some morphological characters that differentiate these species. Adult nematodes collected from the proventriculus of ostriches were observed by light and scanning electron microscopy. Morphologic characterization and morphometric analysis of the nematodes enabled the distinction of both species and corroborated results of prior studies. Specimens of L. dentatus have a buccal capsule with a prominent esophageal tooth. Furthermore, males and females of L. dentatus were larger (4954 and 9347 microm) than those of L. douglassii (3411 and 4229 microm), but measurements for most characters in both species were smaller then those previously reported. Besides, the cephalic structure based on scanning electron microscopy differs, and L. dentatus has thick lips with round papillae, whereas L. douglassii has fine lips with lengthened papillae. The confirmation of both species in South America strongly suggests that the mixed infection may be common in farmed ostriches.

First finding of libyostrongylosis in farm-reared ostriches (Struthio camelus) in Croatia: unusual histopathological finding in the brain of two ostriches, naturally infected with Libyostrongylus douglasi.

In the present work, the very first finding of Libyostrongylus douglasi in farm-reared ostriches in Croatia, was described, not only as the main cause of the death, but also as the cause of persistent infection. The results of histopathological examination of almost all parenchymatous organs in two routinely necropsied ostriches were presented, including atypical histopathological finding in the brain and the result of the L. douglasi scanning electron microscopical (SEM) examination. In order to determine the parasite species to which the larval form found in the brain belonged, polymerase chain reaction (PCR) was performed. Total DNA was isolated from fresh L. douglasi, and from archival formalin-fixed and paraffin-embedded brain section. Additionally, the smears of the proventricular mucosal lining were cytologically examined. Virological examination for newcastle disease virus (NDV) was also performed. As there was very limited information concerned ostrich's health status in Croatian farms, a preliminary evaluation of the parasite infestation level in the Croatian ostrich population over the period 2001-2002 was also done, and an attempt at characterising individual parasite species, was made.

A new species of Sciurodendrium (Nematoda: Heligmonellidae) in Sciurus aureogaster (Rodentia: Sciuridae) from Morelos, Mexico.

Sciurodendrium bravohollisae n. sp. (Heligmonellidae) is described as an intestinal parasite of 2 squirrels, Sciurus aureogaster Cuvier, 1829, collected from Los Robles, Municipio de Tlalnepantla, Morelos State, Mexico. The new species differs from all other congeners in possessing a very well-developed and sacciform genital cone. This is the seventh species described for the genus and the first one recorded in Mexico.

Cuticular Architecture of Hassalstrongylus epsilon (Nematoda: Trichostrongyloidea).

Hassalstrongylus epsilon is a small nematode, whose adult forms are found among the intestinal microvilli of the water rat Nectomys squamipes, Brants 1827 (Rodentia: Muridae). The external appearance of the cuticle, which presents transversal striations and longitudinal ridges, is described using scanning electron microscopy. Transmission electron microscopy of thin sections and replicas of quick-frozen, freeze-fractured, deep-etched and rotatory shadowed samples showed the presence in the cuticle of struts that arise from the fluid median layer, extending outward to the epicuticle. The cuticle showed the presence of five layers: epicuticle, cortical, fibril-rich, fluid median and fibrous. The cuticle layers were made of an assemblage of fibers that create compartments, which were larger in the fluid region than in the fibril-rich median layer.

Nematodirus battus 50 years on--a realistic vaccine candidate?

It has been 50 years since the parasitic nematode of lambs, Nematodirus battus, was first described. This parasite has several interesting features; in particular, it induces a rapid, protective immune response in infected young lambs (< 3 months of age), which is not observed if lambs are infected with other trichostrongyle nematodes. Indeed, protection against most gastrointestinal nematodes only develops once lambs are over five to six months old. In this article I suggest that N. battus offers an opportunity to improve our understanding of protective immune responses in young lambs, and could therefore hold a key to rational anti-nematode vaccine developments, based on natural antigens.

Trichostrongylina (Nematoda) from Malagasy muridae. II--Description of two new species of Heligmonina (Heligmonellidae) in Nesomys rufus and Eliurus tanala.

Two new species of Heligmonellidae from Madagascar are described, Heligmonina madagascariensis n. sp. in Nesomys rufus and Heligmonina tanala n. sp. in Eliurus tanala. Both species belong to the Heligmonina species with a pattern of type 1-3-1 for the right lobe of the caudal bursa and 1-4 for the left lobe. In H. madagascariensis, H. dupuisi (Desset, 1964) and H. praomyos Baylis, 1928, left ray 6 arises before ray 3 from the common trunk to rays 3 to 6 while in H. tanala and the other species, it arises at the same level. H. madagascariensis is differentiated from H. dupuisi and H. praomyos by the symmetry of the branches of the dorsal ray. H. tanala is differentiated from H. malacomys Sakka & Durette-Desset, 1988, the closely related species by a different pattern of the cuticular ridges at mid-body, by the sharpness of the tips of the spicules and by the ratio of the length of the spicules on the length of the body (6.9, 8.8% versus 25-27.8%). Heligmonina chippauxi (Desset, 1964) a parasite of Oenomys hypoxanthus from the Republic of Central Africa is considered a valid species.

Trichostrongylina (Nematoda) from Malagasy Muridae. I--Description of two new species of Heligmonellidae in Nesomys spp.

Two new species of Heligmonellidae, Heligmonina wrightae n.sp. (Nippostrongylinae) and Nesomystrongylus fissicauda n.gen., n. sp. (Brevistriatinae) are described from Madagascar in Nesomys rufus and N. audeberti (Muridae). In Nesomys audeberti, the species are coparasites. Heligmonina wrightae is differentiated from all the other species of the genus, except H. malacomysi Sakka & Durette-Desset, 1988, by the ratio of the length of the spicules on the length of the body (25-27.8% versus 9.5-7%). It differs from H. malacomysi by the pattern of the caudal bursa and by the angle of the axis of orientation of the cuticular ridges on the sagittal axis. Nesomystrongylus fissicauda is related to the genus Fissicauda Durette-Desset & Krishnasamy, 1976, by the absence of the carene, by the ridges discontinuous on all the sides of the body and by the deeply divided dorsal ray. It differs from this genus by a different structure of the ridges, by the pattern of the caudal bursa, (very tiny rays 2 and strongly developed rays 3, rays 8 arising from common trunk of rays 2 to 6) and by the presence of a caudal tip in the female.

On the morphology of Heligmosomoides polygyrus (Nematoda-Trichostrongylidae) from the field mouse apodemus sylvaticus.

The morphology of the trichostrongylid nematode Heligmosomoides polygyrus is described by light and scanning electron microscopy. In light microscope, the present material agrees with the original description given by Baylis (1926), except for the addition of more details about the structure of the genital cone and the synlophe. The scanning electron microscopy of the present nematode revealed that the oral opening of the immature worm (L5) is rounded and its cuticular surface is loosely attached to the body, while the oral opening of the mature worm is triangular, the two copulatory spicules of the male are alate and closely adhering to each other and the right lobe of the male bursa is longer than the left one. Male prebursal and bursal papillae are described. The female posterior extremity is characterized by crescent vulval and anal openings and a terminal spike.

A new host record of Camelostrongylus mentulatus (Nematoda; Trichostrongyloidea) from abomasum of a giraffe at a zoo in Japan.

Camelostrongylus mentulatus (Railliet et Henry, 1909) Orloff, 1933 (Nematoda; Trichostrongyloidea) was found from the abomasum of a three-year-old female cape giraffe, Giraffa camelopardalis giraffa, born and died in a zoo park in Yamaguchi prefecture, Japan. This is the new host record from Giraffidae and geographical distribution of C. mentulatus. Present case of C. mentulatus might be infected from other ruminants, e.g., camels, antelopes and goats, kept at a same paddock in the zoo. Risk of imported parasitic diseases by the zoo animals from outside of Japan is discussed.

Digital image analysis and identification of eggs from bovine parasitic nematodes.

Computer-assisted microscopy and multivariate statistics were used to establish and evaluate a procedure for identification of bovine strongylid eggs. Ostertagia ostertagi, Cooperia oncophora, Haemonchus placei, Trichostrongylus axei, and Oesophagostomum radiatum eggs were obtained from faeces voided by monospecifically infected calves. Images of single eggs (400 x magnification) were recorded by a CCD camera fitted onto a microscope and digitized on a PC. After separation of eggs from the image background, the pixel (picture element) positions of the egg outline were analysed by algorithms to describe size and shape. A stepwise discriminant analysis was subsequently used to select and rank descriptive features of 4207 eggs according to discriminatory power. Classification criteria were developed by linear discrimination analysis on the basis of selected features, and the criteria evaluated by cross-validation. A maximum average percentage of correct classification of 85.8% resulted when nineteen features were employed in a linear classification criterion. The percentages correct classification for each species were: O. ostertagi 76.3%, C. oncophora 90.8%, O. radiatum 87.8%, H. placei 90.1%, and T. axei 83.8%. Classification based on the five most important features gave an overall correct classification of 81.5%. Images of "unknown' eggs could be identified automatically by the classification criteria after procedural steps performed by PC were linked in a batch program.

Use of free living stages to study the effects of thiabendazole, levamisole, pyrantel and ivermectin on the fine structure of Haemonchus contortus and Heligmosomoides polygyrus.

Ultrastructural changes induced in vitro by thiabendazole, levamisole, pyrantel and ivermectin in the free living larval stages of two trichostrongyles (Heligmosomoides polygyrus and Haemonchus contortus) were analysed. The observed damage for each anthelmintic is related to the known mode of action and compared to the damage commonly described in adults. The advantage of using larvae to study the effects of anthelmintics on the fine structure of nematodes rather than adults is described. Thiabendazole induced alteration of the cellular organization especially epithelial cells of the digestive tract. Changes in mitochondria were also seen. Levamisole caused contraction of muscle fibres whereas no specific lesions were observed with pyrantel. Ivermectin caused an hypertrophy of muscular groups. The interest of such a technique in research on the modes of action of anthelmintics is emphasized.

[Oswaldocruzia bonai n. sp. (Nematoda, Trichostrongylina, Molineoidea),a parasite of a Sardinian cave salamander]. / Oswaldocruzia bonai n. sp. (Nematoda, Trichostrongylina, Molineoidea) parasite d'une salamandre cavernicole de Sardaigne.

The Oswaldocruzia parasites of amphibians in Europe are closely related. Contrary to this, the new species Oswaldocruzia bonai discovered in the cave salamander from Sardinia shows particular and archaic characters: dorsal ray of the caudal bursa well developed and tips of the rays 4 equidistant from those of the 3 and 5; female with few eggs of large size. The lack of ridges in front of each lateral cord is a character shared with Ethiopian species but which differentiated the Sardinian species from the European species.