Morphologic and molecular analysis of Dicrocoelium dendriticum species from Iranian native sheep.

Dicrocoeliosis is a worldwide parasitic disease of ruminants which affects the liver. In this current study, the phylogenic pattern of Dicrocoelium species in Iranian native sheep from three different geographical regions was investigated by investigating a 520 bp fragment of mitochondirial NAD1 gene. The analysis of the NAD1 oligo nucleotide sequences from 10 D. dendriticum (GenBank accession numbers: MG889399 to MG889408) revealed few non-significant differences, suggesting limited application for NAD1 gene as a biomarker for study of genetic variation in Dicrocoelium. The morphometrical study also showed a significant relationship for the several morphometric indices among the Dicrocoelium spp. isolates from different regions of Iran.

Vertical Transmission of a Nematode from Female Lizards to the Brains of Their Offspring.

Parasites have evolved a diversity of lifestyles that exploit the biology of their hosts. Some nematodes that parasitize mammals pass via the placenta or milk from one host to another. Similar cases of vertical transmission have never been reported in avian and nonavian reptiles, suggesting that egg laying may constrain the means of parasite transmission. However, here we report the first incidence of transovarial transmission of a previously undescribed nematode in an egg-laying amniote, the common wall lizard (Podarcis muralis). Nematodes enter the developing brain from the female ovary early in embryonic development. Infected lizard embryos develop normally and hatch with nematodes residing in their braincase. We present a morphological and molecular phylogenetic characterization of the nematode and suggest that particular features of lizard biology that are absent from birds and turtles facilitated the evolutionary origin of this novel life history.

Evidence of morphine like substance and µ-opioid receptor expression in Toxacara canis (Nematoda: Ascaridae).

Toxocara canis (Nematoda: Ascaridae) is an intestinal nematode parasite of dogs, which can also cause disease in humans. Transmission to humans usually occurs because of direct contact with T. canis eggs present in soil contaminated with the feces of infected dogs. This nematode has extraordinary abilities to survive for many years in different tissues of vertebrates, and develop to maturity in the intestinal tract of its definitive host. Survival of parasitic nematodes within a host requires immune evasion using complicated pathways. Morphine-like substance, as well as opioids, which are known as down regulating agents, can modulate both innate and acquired immune responses, and let the parasite survives in their hosts. In the present study, we aimed to find evidences of morphine-like substance and µ-opiate receptor expression in T. canis, using high performance liquid chromatography (HPLC) and reverse transcription polymerase chain reaction (RT-PCR). The results indicated that T. canis produced morphine-like substances at the level of 2.31± 0.26 ng g-1 wet weight, and expressed µ-opiate receptor as in expected size of 441 bp. According to our findings, it was concluded that T. canis, benefits using morphine-like substance to modulate host immunity.

The European hedgehog (Erinaceus europaeus), as a reservoir for helminth parasites in Iran.

From April 2009 to December 2011, 44 dead hedgehogs (Erinaceus europaeus) were collected incidentally from areas of Urmia, Iran. The overall prevalence of helminth infections was 95.0%. Specific parasites and their prevalences were: Physaloptera clausa (93.0%), Crenosoma striatum (61.0%), Capillaria aerophila (9.0%), Capillarias spp. (4.0%), Brachylaemus erinacei (2.0%) and Hymenolepis erinacei (16.0%). There were no significant differences in helminth occurrence between hedgehog sexes, either in single or in mixed infections (p > 0.05). The mixed infection involving Crenosoma striatum and P. clausa occurred significantly more frequently than other mixed infection (p < 0.05). There were significant differences in prevalence among seasons, with the highest prevalence in summer and spring especially among P. clausa and C. striatum (p < 0.05).

Hedgehogs (Erinaceus europaeus) as a Source of Ectoparasites in Urban-suburban Areas of Northwest of Iran.

BACKGROUND: Hedgehogs are small, nocturnal mammals which become popular in the world and have important role in transmission of zoonotic agents. Thus, the present study aimed to survey ectoparasite infestation from April 2010 to December 2011 in urban and suburban parts of Urmia and Tabriz Cities, Northwest of Iran. METHODS: A total number of 84 hedgehogs (40 females and 44 males) were examined. They have been carefully inspected for ectoparasites and collected arthropods were stored in 70% ethanol solution. The identification of arthropods was carried out using morphological diagnostic keys. RESULTS: The occurrence of ticks on hedgehogs was 23 (67.7%) with Rhipicephalus turanicus in Urmia and 11 (22%) as well as 1(2%) with Rh. turanicus and Hyalomma anatolicum anatolicum in Tabriz, respectively. One flea species, Archaeopsylla erinacei, was found with prevalence of 19 (55.9%) and 27 (54%) in Urmia and Tabriz Cities, respectively. Prevalence of infestation with Rh. turanicus and A. erinacei were not different (P> 0.05) between sexes of hedgehogs in two study areas. Highest prevalence of tick and flea infestation was in June in Urmia, whereas it was observed in August in Tabriz. Both tick and flea parasitizing hedgehogs showed seasonal difference in prevalence (P< 0.05) in Urmia, but it was not detected in Tabriz (P> 0.05). CONCLUSION: The result showed the high occurrence of ectoparasites in hedgehog population and according to the zoonotic potential of these animals as vector of some agents further studies are needed to investigate in different parts of Iran.

Tegumental ultrastructure of adult Quinqueserialis quinqueserialis (Trematoda: Notocotylidae): an intestinal parasite of muskrat (Ondatra zibethicus).

Ten adult Quinqueserialis quinqueserialis specimens were removed from the intestine of a naturally infected muskrat, and scanning electron microscopy was used to study the morphological characteristics of the trematodes. The mature trematode, which was easy to recognize by the monostome holdfast organ, with no anterior cone, measured 2200-2500 µm in length by 900-1050 µm in width. The body was elongated and tapering at the anterior end, but the posterior end was rounded, and in some specimens was slightly truncated. The mouth opening lay at the anterior end and was surrounded by the oral sucker, which was round, small to medium in size, and subterminal. The tegument of the rim and inside of the oral sucker was smooth and had two types of papillae, domed and rosette papillae. Around the oral sucker, tegument was covered with sharp, pointed spines. The common genital pore was located on the median line of the body, posterior to the oral sucker. The cirrus had smooth tegument at the base and was armed with numerous conical spines throughout its length. The ventral surface was concave and provided with five distinct longitudinal rows of ventral papillae, which extended from the anterior to the posterior end of the body. Each row consisted of 15 to 20 papillae, making 81 to 88 papillae in all. These papillae were variable in size. In most specimens, the papillae were simple knob-like structures, but in some cases, they appeared to be bi- or trifurcate. The tegument at the base of each ventral papilla showed minute spiny pattern, but it was smooth or folded on top and had small rosette and ciliated papillae. Tegument at the edges of the worm was smooth in the mid-parts, spiny on lateral parts, and included rosette papillae. The dorsal surface of the worm was smooth and slightly convex, and the tegument was provided with two large domed papillae in one third of the anterior end of the dorsal part, few thick spines in the mid-part, and excretory pore at the level just posterior to the end. No spines or papillae were seen around the excretory pore.

Molecular systematics of pinniped hookworms (Nematoda: Uncinaria): species delimitation, host associations and host-induced morphometric variation.

Hookworms of the genus Uncinaria have been widely reported from juvenile pinnipeds, however investigations of their systematics has been limited, with only two species described, Uncinaria lucasi from northern fur seals (Callorhinus ursinus) and Uncinaria hamiltoni from South American sea lions (Otaria flavescens). Hookworms were sampled from these hosts and seven additional species including Steller sea lions (Eumetopias jubatus), California sea lions (Zalophus californianus), South American fur seals (Arctocephalus australis), Australian fur seals (Arctocephalus pusillus), New Zealand sea lions (Phocarctos hookeri), southern elephant seals (Mirounga leonina), and the Mediterranean monk seal (Monachus monachus). One hundred and thirteen individual hookworms, including an outgroup species, were sequenced for four genes representing two loci (nuclear ribosomal DNA and mitochondrial DNA). Phylogenetic analyses of these sequences recovered seven independent evolutionary lineages or species, including the described species and five undescribed species. The molecular evidence shows that U. lucasi parasitises both C. ursinus and E. jubatus, whereas U. hamiltoni parasitises O. flavescens and A. australis. The five undescribed hookworm species were each associated with single host species (Z. californianus, A. pusillus, P. hookeri, M. leonina and M. monachus). For parasites of otarids, patterns of Uncinaria host-sharing and phylogenetic relationships had a strong biogeographic component with separate clades of parasites from northern versus southern hemisphere hosts. Comparison of phylogenies for these hookworms and their hosts suggests that the association of U. lucasi with northern fur seals results from a host-switch from Steller sea lions. Morphometric data for U. lucasi shows marked host-associated size differences for both sexes, with U. lucasi individuals from E. jubatus significantly larger. This result suggests that adult growth of U. lucasi is reduced within the host species representing the more recent host-parasite association. Intraspecific host-induced size differences are inconsistent with the exclusive use of morphometrics to delimit and diagnose species of Uncinaria from pinnipeds.

Scanning electron microscopy observations of the hedgehog stomach worm, Physaloptera clausa (Spirurida: Physalopteridae).

BACKGROUND: Physaloptera clausa (Spirurida: Physalopteridae) nematodes parasitize the stomach of the European hedgehog (Erinaceus europaeus) and cause weight loss, anorexia and gastric lesions. The present study provides the first morphological description of adult P. clausa from the stomachs of infected hedgehogs, using scanning electron microscopy (SEM). METHODS: From June to October 2011, 10 P. clausa from European hedgehogs were fixed, dried, coated and subjected to SEM examination. RESULTS: Males and females (22-30 mm and 28-47 mm, respectively) were stout, with the cuticle reflecting over the lips to form a large cephalic collarette and showing fine transverse striations in both sexes. The mouth was characterized by two large, simple triangular lateral pseudolabia, each armed with external and internal teeth. Inside the buccal cavity, a circle of internal small teeth can be observed. Around the mouth, four sub-median cephalic papillae and two large amphids were also observed. The anterior end of both male and female bore an excretory pore on the ventral side and a pair of lateral ciliated cervical papillae. In the female worm, the vulva was located in the middle and the eggs were characterized by smooth surfaces. The posterior end of the female worm was stumpy with two large phasmids in proximity to its extremity. The posterior end of the male had large lateral alae, joined together anteriorly across the ventral surface, with subequal and dissimilar spicules, as well as four pairs of stalked pre-cloacal papillae, three pairs of post-cloacal papillae, and two phasmids. Three sessile papillae occured anteriorly and four posteriorly to the cloaca. CONCLUSIONS: The present SEM study provides the first in-depth morphological characterization of adult P. clausa, and highlights similarities and differences with P. bispiculata P. herthameyerae, Heliconema longissimum and Turgida turgida.

Ultrastructural characterization of male and female Physaloptera rara (Spirurida: Physalopteridae): feline stomach worms.

Physaloptera rara (Spirurida: Physalopteridae) has been found in dogs, coyotes, raccoons, wolves, foxes, cats, and bobcats in North America. The parasites' developmental cycles involve insects, including beetles, cockroaches, and crickets, as intermediate hosts. The nematodes firmly attach to the wall of the stomach and duodenum, where they feed on the mucosa and suck blood. Frequent movement of these nematodes results in erosions and ulcers in the gastrointestinal tract. The present study reports the morphological features of adult P. rara using scanning electron microscopy. Adult worms were removed from the stomach of an infected domestic cat. Male and female worms measured 25-29 and 27-41 mm, respectively. The worms were stout and the cuticle was reflected over the lips to form a large cephalic collarette with fine transverse striations. The worms possessed two large, simple triangular lateral pseudolabia, each armed with one external tooth, three internal teeth, two submedian cephalic papillae, an amphid, and three porous-like circumscribed regions. The internal margins of the lips had a pair of cuticular folds. At the anterior end of both male and female worms, an excretory pore was located on the ventral side and a pair of lateral ciliated cervical papillae was seen. The vulva was anterior to the middle of the body of female worms. The tail ends of the female worms were stumpy, with two large phasmids near their extremities. The males' tails bore large lateral alae. Ventral ornamentation, in male worms, was composed of three different cuticular patterns; coblestone-like formations, longitudinal cuticular ridges, and rows of bead-like structures. The spicules were unequal and dissimilar; the right spicule had a thick end and the left spicule had a sharp tip. At the posterior end of the males, four pairs of stalked precloacal papillae, three pairs of postcloacal papillae, and two phasmids were present. Three and four sessile papillae were seen directly anterior and posterior to the cloaca, respectively. The middle papilla of the three sessile papillae, directly anterior to cloaca was different in shape and size.

New insights into morphological features of Hadjelia truncata (Spirurida: Habronematidae), as revealed by SEM.

Hadjelia truncata (Spirurida: Habronematidae) is a nematode found in the gizzard of several avian species. However, it has been reported pathogenic only in pigeons (Columba livia), in which it causes severe ventriculitis. Scanning electron microscopy (SEM) was used to study the morphological aspects of adult worms of this nematode in detail. Males and females measured 6.5-9 and 12-16.5 mm, respectively. The bodies were straight with moderate uniform thickness in both males and females, narrowing at the anterior end. The cuticle was striated transversely. The mouth was surrounded by two lateral trilobed lips, and the dorsal and ventral interlabia were present. The two cephalic papillae were present on each side lodged just behind the upper and lower bases of each lip. Around the mouth, a large amphid was seen on each lip. At the anterior end of both male and female worms, an excretory pore on the ventral side and a pair of lateral cervical papillae were observed. In the female, the vulva was located at the anterior end of the body. The lips of the vulva protruded above the body surface, and the bluntly rounded posterior end showed an anal pore and two subterminal phasmids. The posterior end of the males curved ventrally, with large caudal alae supported by four pairs of stalked precloacal papillae, a single medial precloacal papilla, two pairs of postcloacal papillae, and a cluster of small papillae on the caudal extremity. Spicules were unequal and dissimilar.

A survey on helminthic infection in mice (Mus musculus) and rats (Rattus norvegicus and Rattus rattus) in Kermanshah, Iran.

Parasitic infections of rodents can compromise scientific research as well as the health of the animals and humans. Based on previous studies, infection rate of parasitic helminths is different in various regions of Iran. The current survey was aimed to determine endoparasitic helminths infection in 138 trapped rodents of Kermanshah county, Iran. Mice and rats were trapped using metal snares from January to October 2011 and euthanized. Rodents included 110 Mus musculus (79.00%), 23 Rattus norvegicus (17.00%), and five Rattus rattus (4.00%). The gastrointestinal and respiratory tracts were removed and examined to identify parasitic helminths. The results indicated that 42.02% of examined rodents were infected with eight helminths species, i.e. Trichuris muris (14.49%), Syphacia obvelata (13.76%), Syphacia muris (2.89%), Aspicularis tetrapetra (5.07%), Heterakis spumosa (5.07%), Capillaria hepatica eggs (3.62%), Hyminolepis diminuta (12.30%), and Cystisercus fasciolaris, the larva of Taenia teanieformis (4.34%). Given the results of this study, we concluded that examined rodents were more infected with nematodes than other helminths. As rodents are usually infected with a number of zoonotic parasites, hence control of these animals has an important role in safeguarding public health.

Ectoparasitic infestations of the European hedgehog (Erinaceus europaeus) in Urmia city, Iran: First report.

Hedgehogs are small, nocturnal mammals that become popular in the world and have significant role in transmission of zoonotic agents. Some of the agents are transmitted by ticks and fleas such as rickettsial agents. For these reason, a survey on ectoparasites in European hedgehog (Erinaceus europaeus) carried out between April 2006 and December 2007 from different parts of Urmia city, west Azerbaijan, Iran. After being euthanized external surface of body of animals was precisely considered for ectoparasites, and arthropods were collected and stored in 70% ethanol solution. Out of 34 hedgehogs 23 hedgehogs (67.70%) were infested with ticks (Rhipicephalus turanicus). Fleas of the species Archaeopsylla erinacei were found on 19 hedgehogs of 34 hedgehogs (55.90%). There was no significant differences between sex of ticks (p > 0.05) but found in fleas (p < 0.05). The prevalence of infestation in sexes and the body condition of hedgehogs (small, medium and large) with ticks and fleas did not show significant differences (p > 0.05). Highest occurrence of infestation in both tick and flea was in June. Among three seasons of hedgehog collection significant differences was observed (p < 0.05). The result of our survey revealed that infestation rate in hedgehog was high. According to zoonotic importance of this ectoparasite and ability to transmission of some pathogens, more studies are needed to investigate hedgehog parasites in different parts of Iran.

Morphological characterization of adult Fascioloides magna (Trematoda: Fasciolidae): first SEM report.

Five adult Fascioloides magna specimens were recovered from the livers of naturally infected calves from Texas, USA. Scanning electron microscopy was used to study the morphological characteristics of the trematodes. These mature flukes measured 35-100 mm in length by 15-25 mm in width and had an oval dorsoventrally flattened body, with no anterior cone. The tegument was armed with sharp spines. Around the oral and ventral suckers, some of the spines were small, with a sharp point, while others had serrated edges with 15-22 sharp points. The surface of the oral sucker was covered by an interesting pattern of tegument, small dome-shaped and ciliated papillae. The ventral sucker showed a smooth surface and two unknown spine-like structures. There were fewer spines at the base of the genital pore than on other parts of the anterior end of the worm. At the anterior end of the ventral side, well-developed spines were observed, while at the posterior end of the ventral side, the spines were small, mostly with one or three points and blunted edges. At the posterior end of the dorsal side, the spines became progressively fewer, smaller, and shorter. Around the excretory pore, the tegument was folded, with no spines, and small groups of dome-shaped and ciliated papillae were present. The cirrus organ showed a smooth surface, with small pores on the dorsal side and small groups of tiny spines between the folds. The eggs measured 168 × 101 µm and had a protoplasmic appendage at the pole opposite the operculum. At the posterior end of the dorsal side, and toward the right, a pore with a very thin rim was present, which could be the terminus of Laurer's canal.

Parasitic ventriculitis caused by Hadjelia truncata in California rock pigeons (Columba livia).

Severe ventriculitis and emaciation caused by the infestation of the nematode Hadjelia truncata occurred in meat-type breeder rock pigeons (Columba livia) in southern and central California. Hadjelia truncata can infest several species of birds, although it has only been reported as pathogenic in pigeons. The factors that contribute to H. truncata pathogenicity are not known. The gross and microscopic pathology caused by the infestation of H. truncata in the ventriculus of pigeons and its morphological identification are presented.

Structural restoration of nematodes and acanthocephalans fixed in high percentage alcohol using DESS solution and rehydration.

Ninety-five percent ethanol is the most widely used field and laboratory preservative for nematodes and other helminth specimens intended for use in molecular systematics. Preservation of nematodes in high-concentration alcohols results in structural dehydration artifacts, including shrinkage and body surface distortions sufficient to obscure features required for morphological identification and analysis, thereby compromising precise morphometrics. However, treating dehydrated nematodes using a solution of DMSO, disodium EDTA, and NaCl, followed by rehydration in water produces marked improvements in specimen shape and surface features, resulting from diffusion of water into the tissues and pseudocoelom as the internal salt concentration is reduced. Following rehydration, tissue samples can be obtained for molecular research and individuals can be fixed for morphological examination. This treatment method is demonstrated for species of 3 nematode genera that vary substantially in body size ( Baylisascaris , Uncinaria , and Bidigiticauda ). The method also works on nematodes that have been cut in half, provided the individuals are large enough to be folded and clamped during treatment. This method appears promising for other helminths, although for an acanthocephalan, the treatment restored the body surface but failed to reverse the retracted proboscis.

An Echinococcus multilocularis coproantigen is a surface glycoprotein with unique O-gycosylation.

A major surface constituent of Echinococcus multilocularis adult worms, referred to as an EmA9 antigen, was immunoaffinity purified and identified as a high-molecular-weight glycoconjugate. Labeling studies using the monoclonal antibody MAbEmA9 indicated that this antigen undergoes a regulated expression during the development from the larval to the adult parasite. Chemical modification of carbohydrate by periodate oxidation resulted in a reduced reactivity with antigen-specific antibodies. Non-reductive beta-elimination of the purified molecule indicated the presence of O-linked glycans attached to threonine residues. Carbohydrate compositional analyses indicated the presence of N- and O-glycans with the ratio of carbohydrate to protein being 1.5:1 (w/w). N- and O-linked glycans were released by hydrazinolysis and analyzed as 2-aminobenzamide derivatized glycans by mass spectrometry together with HPLC and enzymatic sequencing. Novel linear O-linked saccharides with multiple beta-HexNAc extensions of reducing end Gal were identified. N-Linked glycans were also detected with oligomannose and mono-, bi-, tri- and tetra-antennary-type structures, most of which were found to be core-fucosylated. Taken together, the results indicate that the EmA9 antigen is a glycoprotein located at the outer surface of the adult E. multilocularis. The observation that the EmA9 antigen expression is developmentally regulated suggests an involvement of this glycoprotein in the establishment of the parasite in its canine host.

The comparative morphology of three equine habronematid nematodes: SEM observations.

Drashia megastoma Rudolphi, 1819, Habronema muscae Carter, 1861 and Habronema microstoma Schneider, 1866 are found in the stomach of equine definitive hosts and are known to cause pathogenic effects in the stomach wall, skin, eye and occasionally other sites. These nematodes utilise either house flies or stable flies as their intermediate hosts. Apart from molecular findings that have demonstrated some of the differences between H. muscae and H. microstoma, no detailed morphological description of equine habronematid nematodes has been presented. This article describes most surface features of adult D. megastoma, H. muscae and H. microstoma using scanning electron microscopy and differentiates these nematodes based on morphological characteristics.

Equine stomach worm, Drashia megastoma (Spirurida: Habronematidae): first SEM report.

Drashia megastoma (Spirurida: Habronematidae) occurs in nodules in the stomach wall and rarely free in the stomach of the horse, mule and zebra throughout the world. D. megastoma develops in the housefly Musca domestica and causes gastric haemorrhage or even perforation of the stomach. Larvae of this nematode may be found in the lung, skin and eye of the host. Scanning electron microscopy (SEM) was used to study the surface ultrastructure of adult worms of this nematode. In both sexes, the head end was separated from the rest of the body by a constriction. The stoma was infundibuliform with a wall forming two separated lateral vales. The pseudo-labia were not lobed. Around the mouth, four submedian cephalic papillae and two amphids were seen. A pair of ciliated cervical papillae was present, and an excretory pore was observed. There were two lateral alae. In the female, the vulva was opened anteriorly. The caudal end of the male was alate and the spicules were unequal. At the posterior end of the male, four pairs of stalked precloacal papillae, two median precloacal papillae close to the cloaca, two pairs of postcloacal papillae and a cluster of small papillae were present.

First SEM observations on adult Habronema microstoma (Spirurida: Habronematidae), a parasite of the horse.

Habronema microstoma (Spirurida: Habronematidae) is found in the stomach of equines and uses the stable fly as its intermediate host. This nematode causes pathogenic effects in the stomach wall, skin and eye of the host. The present study reports the morphological features of adult worms, using scanning electron microscopy (SEM). The worms had two bilobed lateral lips surrounding the mouth. The buccal vestibule was markedly thickened, and two tridentate teeth were observed. Around the mouth, four sub-median cephalic papillae and two amphids were seen. A pair of lateral cervical papillae was present. There was only one lateral ala in both sexes. In the female, the vulva was opened in the middle of the body. In the male, there were wide caudal alae, and the spicules were unequal. At the posterior end of the male, four pairs of stalked pre-cloacal papillae, a single lateral pre-cloacal papilla, two pairs of post-cloacal papillae and a cluster of small papillae were present.

First description of the horse stomach worm, Habronema muscae (Spirurida: Habronematidae) by scanning electron microscopy.

Habronema muscae (Spirurida: Habronematidae) occurs in the stomach of equids, is transmitted by adult muscid dipterans and causes gastric habronemiasis. Scanning electron microscopy (SEM) was used to study the morphological aspects of adult worms of this nematode in detail. The worms possess two trilobed lateral lips. The buccal cavity was cylindrical, with thick walls and without teeth. Around the mouth, four submedian cephalic papillae and two amphids were seen. A pair of lateral cervical papillae was present. There was a single lateral ala and in the female the vulva was situated in the middle of the body. In the male, there were wide caudal alae, and the spicules were unequal and dissimilar. At the posterior end of the male, four pairs of stalked precloacal papillae, unpaired post-cloacal papillae and a cluster of small papillae were present. In one case, the anterior end showed abnormal features.