Spirocerca lupi draft genome, vaccine and anthelmintic targets.

Spirocerca lupi is a parasitic nematode affecting predominantly domestic dogs. It causes spirocercosis, a disease that is often fatal. The assembled draft genome of S. lupi consists of 13,627 predicted protein-coding genes and is approximately 150 Mb in length. Several known anthelmintic gene targets such as for ß-Tubulin, glutamate, and GABA receptors as well as known vaccine gene targets such as cysteine protease inhibitor and cytokines were identified in S. lupi by comparing orthologs of C. elegans anthelmintic gene targets as well as orthologs to known vaccine candidates. New anthelmintic targets were predicted through an inclusion-exclusion strategy and new vaccine targets were predicted through an immunoinformatics approach. New anthelminthic targets include DNA-directed RNA polymerases, chitin synthase, polymerases, and other enzymes. New vaccine targets include cuticle collagens. These gene targets provide a starting platform for new drug identification and vaccine design.

Subcutaneous spirocercosis in a dog from Costa Rica and first molecular confirmation of Spirocerca lupi in Central America.

Spirocerca lupi is the etiological agent of spirocercosis in canids from tropics and subtropics in the world. This parasite can lead to life threatening complications, including the formation of fibro and osteosarcomas. A 5-year-old crossbreed Maltese dog from Costa Rica was presented to a veterinary clinic with two nodules in the subcutaneous tissues of the cervical region. One of the nodules resolved with intramuscular antibiotic and anti-inflammatory treatment. However, the other nodule persisted and became purulent. The content of the nodule was excised releasing two coiled 3-cm reddish worms. Identity of the specimens was confirmed by amplifying an 850 bp fragment of the 18S rDNA. The obtained sequence showed 99.96% similarities with S. lupi from Hungary. The present study highlights the need of veterinary clinicians' awareness towards nematode aberrant migrations, and the require of molecular studies to gain insight into the parasite's evolutionary history.

Identification of the intermediate host of Gongylonema sp., the etiological agent of the necrotic oropharyngeal disease of the Scops owl (Otus scops).

Since 1997, fledgling Scops owls (Chordata: Strigidae) have been brought to the Brinzal Owl Rescue Centre (Madrid, Spain) with severe lesions in their oral cavities. Lesions consist of the presence of proliferative necrotic material in the oral cavity resulting in white plaques, which can lead to death by starvation. This disease has been detected in owls only within the limits of the city of Madrid. The etiologic agent has been identified as Gongylonema sp. (Nematoda: Spirurida), a nematode genus that includes a coprophagous arthropod as intermediate host in its cycle. The aim of this study was to identify the intermediate host of the parasite. Our work was structured in four component phases: i) Diet study of newborn chicks; ii) trapping arthropods that could be intermediate hosts; iii) molecular detection of the parasite in the selected arthropods: and iv) molecular characterization of the detected parasites by amplifying the cox1 gene. Four male owls were radio-tagged in order to locate their nests and a camera trap was placed to identify the prey brought to the owlets. Secondly, the arthropods present in the hunting areas of the owls were sampled, identified and analyzed by real time PCR (rtPCR). Only oriental cockroaches, B. orientalis (Arthropoda: Blattodea), were positive by rtPCR detection of Gongylonema sp. (66.7%). The nematodes obtained from cockroaches had a 99.8% identity of the cox1 gene with the Gongylonema sp. isolated for the first time in a Scops owl. Furthermore, these sequences only showed an <89% identity with all the other Gongylonema sequences available in the GenBank database. We conclude that the oriental cockroach should be considered as an intermediate host of the etiologic agent of NOD.

Morphological and molecular characterization of Spirocerca lupi (Nematoda: Spirocercidae) from domestic dogs in Vietnam with reference to Spirocerca vulpis.

Spirocerca lupi is a common parasitic nematode associated with esophageal cancer of canids. Recent surveys have revealed an increasing number of canids infected with Spirocerca spp. in Africa, the Americas, Europe and Western Asia, and described a new species, Spirocerca vulpis, from red foxes (Vulpes vulpes). However, in Southeast Asia, research on Spirocerca spp. is scarce. Therefore, the aim of this study is to explore Spirocerca infection in domestic dogs in Vietnam and to identify the Spirocerca species by analyzing their morphometric and molecular data. We found Spirocerca spp. specimens in 51 (17.7%) out of 287 dogs examined with the intensity of infection ranging from one to 29 nematodes per dog. These nematodes were morphologically and molecularly identified as S. lupi. For morphology, the presence/absence of teeth, the ratio of glandular to muscular esophagus lengths, and the position of the vulva opening of S. lupi and S. vulpis varied between reports, suggesting caution in identification of Spirocerca species based exclusively on morphological characteristics. The molecular analysis based on a partial cox1 sequence revealed that S. lupi from Vietnam is genetically close to those from India and China, but far different from those of Israel, South Africa, Peru and Hungary. Given high genetic and morphological variations, more extensive surveys on Spirocerca spp. from various mammalian hosts at a greater scale are necessary to elucidate the divergence of this nematode.

Central nervous signs, blindness and cerebral vermicosis in free-ranging peregrine falcons (Falco peregrinus) associated with aberrant larval migrations.

Four free-ranging peregrine falcons (Falco peregrinus) were submitted with a history of unilateral or bilateral blindness and central nervous signs to a veterinary clinic in Germany. There were no indications of trauma or ocular disease. Likewise, other differential diagnoses for CNS signs were ruled out within the diagnostic process. The clinical diagnostic panel in live falcons included general examination, radiography, endoscopy, hematology, ophthalmoscopy and parasitological examination of the feces, blood gas analysis and blood chemistry as well as computed tomography, and magnetic resonance imaging (MRI). A complete pathological and histopathological examination was performed post-mortem. The only common finding in all birds was an infection with the nematode parasite Serratospiculum tendo. The parasite was confirmed morphologically and via PCR. In two falcons intracerebral vermicoses was suspected in MRI and confirmed in subsequent histopathology, but molecular biological identification of the parasite species failed from brain tissue. Until today, S. tendo had been reported to affect the respiratory system, the liver and different parts of the gastrointestinal tract and to cause cachexia, inappetence, regurgitation, dyspnea and general signs of illness such as lethargy, poor plumage, and reduced reproduction. Our findings indicate that aberrant migration should be considered as cause for CNS signs in falcons. As S. tendo might be a possible cause for this, CNS signs might be included in the list of clinical signs of serratospiculiasis in falcons.

Secretome of the carcinogenic helminth Spirocerca lupi reveals specific parasite proteins associated with its different life stages.

Spirocerca lupi is a parasitic and carcinogenic nematode of canids distributed in tropical and subtropical regions around the world. The excretion-secretion proteins (PES) of S. lupi have been suggested to play a role in the pathogenesis of its infection. We aimed to identify the PES of different stages of S. lupi and search for proteins that would be useful for diagnostic, therapeutic and vaccination purposes as well as understand their functions. A nano-UPLC mass spectrometry de novo analysis was performed on proteins collected from cultures of S. lupi L3 larvae, L4 females, adult females and adult males from naturally infected hosts. A total of 211 proteins were identified in all cultures. Accordingly, 117, 130, 99 and 116 proteins were detected in L3 larva, L4 females, adult females and adult males, respectively, with a strong correlation in the biological replicates (Pearson coefficients > 0.73). Fourty-four proteins were detected in all developmental stages, 64 were stage-specific and 49 were exclusively identified in L4 females. Cell compartment enrichment analysis revealed that proteins common to all stages were cytoplasmatic (p < 9.x10-6), whereas L4 unique proteins were in collagen trimers, and macromolecular complexes (p < 0.00001). Functional enrichment analysis of proteins showed significant enrichment in lipid metabolism in L3-unique proteins (p<0.00005), in mannose metabolism and protein de-glycosylation for L4-unique proteins (p < 0.00004), and in phosphorus metabolism in proteins shared by all stages (p <  2.1 x10-9). Interestingly, annexin 6, associated with cancer in humans, was detected in all life stages, but in a larger abundance in L4 females and adults. These findings indicate that S. lupi establishes complex interactions with its hosts by an arsenal of proteins expressed in different patterns in each life stage which influence the pathogenesis and oncogenesis of S. lupi and may be used as potential targets for diagnostic assays, drug targets or vaccine candidates.

Incidence of cutaneous habronemosis in Manipuri ponies in India.

Information pertaining to parasitic fauna and parasitic diseases in Manipuri ponies in India is not available. Moreover, no systematic studies have been undertaken on cutaneous habronemosis in Manipuri ponies which is a common skin problem of Manipuri ponies as reported by pony owners. Keeping in the view of the importance of parasitic infections in veterinary health coverage particularly in Manipuri ponies, the present study was planned. A survey of natural cases of cutaneous habronemosis followed by molecular confirmation of species involved and treatments were done. Out of 200 ponies examined, nine cases (4.5%) of cutaneous habronemosis was recorded. Gross examination revealed raised and ulcerated wounds with necrotic tissues covered with yellowish-tan granulation. Histopathological study revealed eosinophilic granuloma and in the center of the granuloma with necrotic debris. Remnants of the Hebronema larvae with infiltrating neutrophils surrounded by proliferating fibrous tissue with numerous eosinophils, macrophages and lymphocytes were also observed. Molecular detection of Habronema sp. was confirmed by semi-nested PCR. Sequence analysis revealed larvae of H. muscae was the common spirurid species responsible for producing cutaneous habronemosis in Manipuri ponies. Subsequently, sequence submitted to NCBI GenBank and accession number obtained (MH038181). Surgical removal of necrotic tissue, ivermectin injection along with antibiotics successfully cured all the lesions in infected ponies.Results confirmed occurrence of cutaneous habronemosis in Manipuri ponies in India.

A New Species of Ascarophis (Nematoda: Cystidicolidae) Parasitizing Clinocottus analis (Pisces: Cottidae) from Baja California, Mexico.

A new species of nematode, Ascarophis morronei n. sp. (Cystidicolidae), is described from the stomach wall of the woolly sculpin Clinocottus analis (Cottidae) collected in the rocky intertidal from northwestern Baja California, Mexico. Collected nematodes were studied using both light and scanning electron microscopy. Sequence fragments for 18S rDNA molecular markers were obtained from the new nematode species, in order to test its position within the family Cystidicolidae under a phylogenetic context. Main characters distinguishing this new species include the reduced labia and the morphology of the eggs, distances of nerve ring and excretory pore from the anterior end, and left spicule of males. The new species described here is the second for the genus Ascarophis reported as adult in the Southern California Bight, and the first one recorded for the fish genus Clinocottus.

The first autochthonous case of feline ocular thelaziosis in Austria.

Over the last 30 years, Thelazia callipaeda (Spirurida: Thelaziidae) has increasingly been reported as an agent of ocular infections in animals and humans throughout Europe. Following the cases of canine ocular thelaziosis recently recorded in Austria for the first time, in the present paper, we describe the first case of T. callipaeda infection in an Austrian cat with no history of traveling abroad. This finding further supports the occurrence of the parasite's autochthonous transmission cycle in the country. The cat showed serous ocular discharge, conjunctival hyperemia, and mild conjunctival edema in the right eye. Mechanical removal of the parasite from the cat's eye, in combination with milbemycin oxime/praziquantel oral treatment and topical use of tobramycin/dexamethasone eye drops led to complete resolution of the clinical signs within 2 weeks. Results presented in the current study are of great importance for the local veterinarians who seemed largely unaware of this zoonotic parasite. Therefore, increased awareness of medical and veterinary communities is imperative for preventing further infections in both animals and humans.

Periocular thelaziasis presenting as an orbital mass - a case report.

Thelazia callipaeda is a rare parasitic infestation caused by spiruroid nematode of the genus Thelazia. We report a case of a 74-year-old gentleman who presented with a painless swelling of left lower lid since 15 days. Examination revealed a firm mobile mass along the inferior orbital rim. Magnetic Resonance Imaging showed a well-defined preseptal cystic lesion and Ultrasound screening revealed multiple mobile worms within. Patient underwent cyst excision in toto under local anesthesia. Four long refractile worms were isolated from within the cyst cavity. Species identification confirmed the parasite as Thelazia callipaeda. Periocular thelaziasis usually presents as free floating worms in the conjunctival sac, anterior chamber or vitreous cavity. It is important to be aware of this rare entity which should be considered as a differential diagnosis in endemic areas.

Rhabdochona angusticaudata sp. n. (Nematoda: Rhabdochonidae) from the Japanese eel Anguilla japonica, and new records of some other nematodes from inland fishes in Japan.

Collections of parasitic nematodes of fishes from some freshwater and brackish-water localities in Honshu, Shikoku and Hokkaido, Japan, carried out in 1996-2009, yielded the following eight species: Hysterothylacium haze (Machida, Takahashi et Masuuchi, 1978) (Anisakidae), Paraquimperia sp., Pingus sinensis Hsü, 1933 (both Quimperiidae), Heliconema anguillae Yamaguti, 1935 (Physalopteridae), Rhabdochona angusticaudata sp. n., Rhabdochona zacconis Yamaguti, 1935 (both Rhabdochonidae), Ascarophis arctica Polyanskiy, 1952 (Cystidicolidae) and Eustrongylides sp. larvae (Dioctophymatidae). The new species, R. angusticaudata sp. n., described from the intestine of the Japanese eel Anguilla japonica Temminck et Schlegel in the Renjoji River, Shikoku, is mainly characterised by the presence of 14-16 anterior prostomal teeth, non-bifurcated deirids with rounded ends, the length of the left spicule (300-384 µm long), the right spicule with a dorsal barb and non-filamented eggs. The species of Paraquimperia Baylis, 1934 and A. arctica are reported from Japan for the first time. Based on light and scanning electron microscopical examinations, some new morphological data on H. haze, H. anguillae, R. zacconis and A. arctica are provided.

Live encysted Thelazia callipaeda presenting as a lump adjacent to the right lacrimal sac in a 42-year-old female: A rare case report.

Thelazia callipaeda or the oriental eye worm infects a wide variety of hosts including dogs, cats, foxes, rabbits, and humans through the Drosophila flies. We report here a case of a 42-year-old female who presented with pain, redness, and cystic swelling in the right lacrimal sac area for 8 months. Ocular examination was within normal limits. Excision biopsy of the cyst revealed a live worm. In humans, the worm is usually found in the conjunctival sac, lacrimal canaliculi, or the lacrimal sac leading to a disease which can be subclinical or symptomatic. Thus, we report a rare presentation of thelaziasis, that is, periocular involvement. This case presentation is first of its kind in the Indian literature.

Acute phase proteins, interleukin-6, tumor necrosis factor, nitric oxide and oxidative stress markers in horses with cutaneous habronemosis under field condition.

Habronemosis is a common parasitic disease of horses worldwide. In order to investigate how haptoglobin (Hp), serum amyloid A (SAA), oxidative stress markers, nitric oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor (TNF- α), varies in cutaneous habronemosis, 30 horses with the clinical disease and 20 clinically healthy horses were included in the current study. The serum levels of Hp, SAA, and proinflammatory cytokines (IL-6 and TNF-α), NO, malondialdehyde (MDA), super oxide dismutase (SOD), glutathione (GSH), and total antioxidant capacity (TAC) were determined in horses before and after two weeks of treatment. The serum levels of Hp, SAA, IL-6, TNF-α and MDA were significantly elevated in infected horses as compared to the controls. Alternately, the serum levels of SOD, GSH, TAC and NO, were recorded low in infected horses as compared to the controls. All tested markers resumed the same levels after treatment as in control group. The Hp, SAA, IL-6, TNF-α, and MDA exhibited a high degree of clinical accuracy of the cases diagnosis. The area under the curve (AUC) for acute phase proteins (SAA, Hp), IL-6, TNF-α, and MDA was 0.87, 0.94, 0.96, 0.96 and 1.0, respectively. These findings showed that Hp, SAA, IL-6, TNF-α, and MDA may be supportive in the diagnosis of cutaneous habronemosis in horses and, simultaneously, they can also be used to monitor the progress of the treatment.

Two nematode species from freshwater and marine fishes in Thailand, including Ascarophis scatophagi sp. nov. (Cystidicolidae) from Scatophagus argus (Scatophagidae).

One new and one known species of parasitic nematodes are reported from Thailand for the first time: Falcaustra kaverii (Karve et Naik,1951) (Kathlaniidae) from the intestine of the freshwater fish Poropuntius deauratus (Valenciennes) (Cyprinidae) (new host record) and Ascarophis scatophagi sp. nov. (Cystidicolidae) from the intestine of the marine fish Scatophagus argus (Linnaeus) (Scatophagidae). The morphology of both these species was studied with the use of light (LM) and scanning electron microscopy (SEM) (the latter method used for F. kaverii for the first time). The new species (A. scatophagi) is mainly characterized by nonfilamented, markedly elongate-oval eggs (size 42-45 × 18 µm), spicules 555-642 µm and 105-150 µm long, the muscular/glandular oesophagus length ratio (1:13-34), bifurcate deirids, the length of the female tail (159-249 µm) and cephalic structures typical of the nominotypical subgenus Ascarophis. Representatives of Falcaustra Lane, 1915 and Ascarophis van Beneden, 1871 were not previously reported from fishes in Thai waters.

Redescription and genetic characterization of Cystidicoloides vaucheri, including first description of male and current status on the phylogeny of Cystidicolidae (Nematoda: Habronematoidea).

Cystidicoloides vaucheri collected in the stomach of the redtail catfish Phractocephalus hemioliopterus from River Acre, State of Acre, Brazil is redescribed, including the first description of males and the first genetic characterization based upon 18S and 28S genes of the rRNA. Newly collected females were biometrically smaller than those reported in the original description, but similar morphology shared by the two samples revealed that they belong to the same species. Scanning electron micrographs showed the accurate structure of the cephalic region, described here in detail. Furthermore, the morphology of males completed the specific diagnosis, strengthening the validity of the species. The three other congeners differ from C. vaucheri mainly as follows: in C. dlouhyi the area rugosa is absent, the cephalic structures in C. fischeri are completely distinct, and in both species the spicules have membranous outgrowths, absent in C. vaucheri. Despite the dubious generic assignment of C. izecksohni, it differs from C. vaucheri in several biometrical and morphological features. Because of data availability, only sequences of the 18S were used for phylogenetic reconstructions. Results showed that the genus Ascarophis and the families Cystidicolidae and Physalopteridae are not monophyletic. Cystidicoloides vaucheri formed an independent branch clustering with representatives of Cystidicolidae, confirming its validity. The inclusion of Salmonema and Spinitectus within Cystidicolidae should be reviewed, since they formed an assemblage with species from Rhabdochonidae. In fact, current classification of some taxa belonging to Habronematoidea, Physalopteroidea and Thelazioidea need to be re-evaluated, mainly based on molecular data from different genes.

Gongylonema Parasites of Rodents: A Key to Species and New Data on Gongylonema neoplasticum.

Specimens of the genus Gongylonema were collected from the gastric mucosa of rodents of Rattus rattus Linnaeus, 1758, and Rattus norvegicus Berkenhout, 1769, collected in urban areas in Belém, Pará, in the eastern Brazilian Amazon. The helminths were processed for analysis using light microscopy and scanning electron microscopy (SEM) techniques and presented taxonomic characteristics of the species Gongylonema neoplasticum. The SEM analyses revealed the presence of 2 developed buccal plates (1 dorsal, 1 ventral), also called interlabia, with a prominent and bifurcated ventral plaque. The occurrence of the bifurcated ventral interlabium had not yet been identified by any other author from G. neoplasticum. As a result of our extensive research on published data on Gongylonema spp., we propose a taxonomic key for species of this genus that parasitize rodents. This is the first record of G. neoplasticum in urban areas of the Brazilian Amazon.

An adult cystidicolid nematode (Nematoda: Cystidicolidae) from the subcutaneous tissue around the eye of percupinefish, Diodon nichthemerus Cuvier.

Three adult nematode specimens, all ovigerous females belonging to the family Cystidicolidae Skryabin, 1946, were found for the first time in the subcutaneous tissue around the eye of the captive porcupinefish Diodon nichthemerus Cuvier at a public aquarium in Osaka, Japan. Because no male was available, these could not be identified to the genus and species. This case highlights the risk of parasitism in aquaculture puffer fish, as these may ingest small shrimp, which probably act as intermediate hosts for the nematode.

In vitro lectin binding to the outer surface of Spirocerca lupi at different life-stages.

Spirocerca lupi is the esophageal nematode of dogs. Early, transient eosinophilia occurs in experimentally infected dogs, but is absent in advanced cases, suggesting that the nematode evades the dog's immune system. Lectins are proteins or glycoproteins of plant or animal origin, binding different saccharides, with varying specificities and avidities, used to characterize surface haptens in plant and animal parasitic helminths. This study investigated the in vitro binding of six lectins (Concanavalin A [ConA], wheat germ agglutinin [WGA], peanut agglutinin [PNA], soybean agglutinin [SBA], Dolichus biflorus agglutinin [DBA] and Ulex earopaeus agglutinin I [UEA]) to the surface of S. lupi nematodes at different life stages, the L2 and L3 larvae (dead and alive) and to dead adult worms, with negative controls, with and without addition of the six respective inhibitory sugar haptens. Con A moderately bound to surfaces of both live and frozen L3, to the stoma and excretory pores of adult worms, and to the outer surface nematode's eggs, within a female worm, but not to L2. PNA bound only to stoma and excretory pores surfaces in both frozen and live L3. WGA bound strongly to the outer surfaces of live and dead L2 and L3, which resulted in molting of live larvae. These results suggest that the nematode's surface content change during its development. Such changes may play roles in the nematode's interactions with the intermediate and definitive hosts' tissues, and in its ability to evade the immune response, its long survival within the host, and even induce neoplastic transformation.

Redescription of two species of cystidicolid nematodes (Spirurina: Cystidicolidae) from Notopterus notopterus (Osteichthyes) in Thailand.

Two nematode species, Pseudoproleptus notopteri (Karve et Naik, 1951) and Spinitectus notopteri Karve et Naik, 1951 (both Cystidicolidae), are redescribed based on light and scanning electron microscopical studies of specimens collected from the digestive tract of the freshwater fish Notopterus notopterus (Pallas) (Notopteridae, Osteoglossiformes) in Thailand. Some new important morphological features, such as a detailed structure of the cephalic end and the presence of bifurcate deirids and a ventral median caudal protuberance in male, are reported for the former species (P. notopteri), which is provisionally assigned to Pseudoproleptus Khera, 1955; Notopteroides notopteri Chakravarty et Majumdar, 1962, Pseudoproleptus satendri Sahay, 1967, P. lamyi Le-Van-Hoa et Bui-Thi Lien-Huong, 1969, P. gomtii Gupta et Bakshi, 1984. P. sprenti Gupta et Masoodi, 1986 and P. thapari Gupta et Naiyer, 1992 are considered its junior synonyms. The first study of S. notopteri by SEM showed its morphological similarity with S. mastacembeli Karve et Naik, 1951, from which it clearly differs by the structure of eggs; Spinitectus alii Kalyankar, 1970, S. bengalensis Chakravarty, Sain et Majumdar, 1961, S. gomalensis Siddiqui et Kattak, 1984 and S. thapari Ali, 1957 are considered to be junior synonyms of S. notopteri. Pseudoproleptus notopteri and Spinitectus notopteri are reported from Thailand for the first time.