The use of primary murine fibroblasts to ascertain if Spirocerca lupi secretory/excretory protein products are mitogenic ex vivo.

BACKGROUND: Spirocerca lupi is a nematode that parasitizes vertebrates in particular canids, by forming nodules in the thoracic cavity specifically in the oesophagus. In 25% of Spirocerca infections of the domestic dog, nodules progress from inflammatory to pre-neoplastic to sarcomatous neoplasia. With the mechanism of neoplastic transformation being incompletely understood, this study investigates if S. lupi parasite proteinaceous secretory/excretory products (ESPs) play a role in the neoplastic transformation. METHODS: To facilitate collection of ESPs, we maintained naturally harvested adult parasites in the laboratory under artificial conditions. Media in which the parasites were grown was subsequently evaluated for the presence of proteinaceous compounds using a mass spectroscopy library as well as for their ability to be mitogenic in primary murine fibroblastic cells. RESULTS: Chromatrography of the ethyl acetate extracted incubation media showed the presence of 9 protein compounds, of which three were identified as non-specific proteins isolated from Nematostella vectensis, Caenorhabditis brenneri and Sus scrofa, with the rest being unknown. Acetone, methanol, hexane and ethylacetate extracted culture media were unable to induce a mitogenic change in primary murine fibroblasts in comparison to the controls. CONCLUSION: While no mitogenic effect was evident, further studies are required to understand the role of worm excretory/secretory products on clastogenesis under chronic exposure. In addition, while not of primary importance for this study, the observed duration of parasite survival indicates that ex vivo studies on S. lupi are possible. For the latter we believe that the worm culture method can be further optimized if longer survival times are required.

Genotyping and comparative pathology of Spirocerca in black-backed jackals (Canis mesomelas) in South Africa.

BACKGROUND: The pathology of spirocercosis, a disease caused by the infestation of carnivores with the nematode Spirocerca lupi, has been extensively described in domestic dogs and coyotes. However, it has not been described in wild carnivores in South Africa. The aim of this study was to evaluate whether black-backed jackals are a host for Spirocerca species and to provide a detailed description of the associated pathology. Jackals were also stratified according to age and the Spirocerca species recovered were characterized using molecular techniques. METHODS: Standard necropsies were performed on routinely culled jackals from three of the nine provinces of South Africa during the period June 2012 to February 2013. Jackals were screened for the presence of pathognomonic Spirocerca-induced lesions and for evidence of aberrant migration. Relevant samples were submitted for histopathology and collected larvae were genotyped at nine microsatellite loci. RESULTS: Spirocerca lupi-associated aortic lesions were found in 16 of 93 (17%) black-backed jackals. Of these, four (25%) were associated with S. lupi larvae. Genotyping of the larvae revealed amplification of all nine loci that amplified dog-derived S. lupi, with the same level of polymorphism in the allele size ranges. Only 1 of 93 jackals had an esophageal nodule with concurrent S. lupi-induced aortic aneurysms. The single esophageal nodule found did not contain adult nematodes, nor did it communicate with the esophageal lumen. None of the jackals that were examined had macroscopically evident spondylitis, which is frequently reported in the dog. Histopathology of the S. lupi-induced aortic lesions in the jackal revealed replacement of elastic and smooth muscle fibers by fibrous connective tissue. In cases where inflammation was present, the inflammatory infiltrate consisted predominantly of eosinophils. The single esophageal nodule histologically resembled the early inflammatory nodule described in dogs and consisted of fibrous connective tissue, multifocal accumulation of lymphocytes, plasma cells and rare hemosiderin-laden macrophages. CONCLUSIONS: These lesions suggest that the life cycle of S. lupi may not or only rarely be completed in jackals. A possible explanation might be that jackals are relatively resistant to developing significant pathology associated with S. lupi-infection. However, before any conclusions can be drawn, many more jackals, including those that die naturally will have to be investigated for evidence of S. lupi infection.

Development of some larval nematodes in experimental and natural animal hosts: an insight into development of pathological lesions vis-a-vis host-parasite interactions.

Infective third-stage larvae of three spiruroid nematodes, Ascarops strongylina and Physocephalus sexalatus of pigs and Spirocerca lupi of dogs, were recovered from 14 species of coprophagous beetles belonging to 4 different genera. These larvae were fed to rabbits and/or guinea pigs to study their development in these experimental hosts. Larvae of A. strongylina reached the adult stage in all rabbits and one guinea pig. The adult worms recovered in these hosts were 40% and 4%, respectively, and became diminutive in comparison to their natural hosts. The larvae of P. sexalatus became reencysted in the gastric wall of rabbits inducing marked pathological changes. The infective larvae of S. lupi became reencapsulated in the stomach wall of the rabbit and also showed development in the aortic wall. Adults of Toxocara canis of dog, collected from 5 different regions of the Indian subcontinent, varied significantly in size. The mouse passage of infective larvae of one of these types led to the recovery of the adults from the experimental dogs that were smaller in size and caused severe pathology in natural experimental hosts. Developmental effects shown in experimental hosts and host specificity are of value in understanding the evolution of nematode parasitism.

Immunohistochemical characterization of lymphocyte and myeloid cell infiltrates in spirocercosis-induced oesophageal nodules.

Spirocerca lupi is a nematode that infects the dog's oesophagus and promotes the formation of an inflammatory fibroblastic nodule that progresses to sarcoma in approximately 25% of cases. Spirocercosis-associated oesophageal sarcoma is an excellent and under-utilized spontaneous model of parasite-associated malignancy. The inflammatory infiltrate of paraffin-embedded, non-neoplastic oesophageal nodules (n = 46), neoplastic nodules (n = 25) and normal oesophagus (n = 14) was examined by immunohistochemistry using MAC387 (myeloid cells), CD3 (T cells), Pax5 (B cells) and FoxP3 (T regulatory cells) antibodies. Myeloid cells predominated in 70% of nodules, in pockets around the worms' migratory tracts and in necro-ulcerative areas in neoplastic cases. T cells predominated in 23% of cases with a focal or diffuse distribution, in the nodule periphery. No significant differences were observed between neoplastic and non-neoplastic stages. FoxP3+ cells were observed in low numbers, not significantly different from the controls. The inflammation in spirocercosis is characterized by pockets of pus surrounded by organized lymphoid foci. There was no evidence of a local accumulation of FoxP3+ cells, unlike many previous studies that have reported an increase in FoxP3+ T cells in both malignancies and parasite infections. The triggering factor(s) driving the malignant transformation of the spirocercosis-associated chronic inflammatory nodule warrants further investigation.

Experimental infection of selected arthropods with spirurid nematodes Spirocerca lupi Railliet & Henry, 1911 and Gongylonema ingluvicola Molin, 1857.

Gongylonema ingluvicola and Spirocerca lupi are spirurid nematodes that require arthropod intermediate hosts in order to complete their life cycle. Beetles of the family Scarabaeidae are reported to serve as intermediate hosts for both these parasites. In this study selected species of beetles of the family Scarabaeidae as well as other groups of arthropods were screened for susceptibility to infection with S. lupi and G. ingluvicola. Arthropods were exposed to infective eggs of both parasites for a determined period of time and dissected/digested to determine the presence or absence of pre-infective and infective larvae. All the five species of dung beetles exposed to infection with S. lupi, namely, Pachylomerus femoralis, Scarabaeus rugosus, Gymnopleurus humanus, Kheper nigroaeneus and Anachalcos convexus were susceptible and, of the two species exposed to G. ingluvicola, only Gy. humanus was susceptible. Spirocerca lupi eggs developed in millipede species, Daratoagonus cristulatus, and remained as encysted larvae, while in Orthoporoides kyrhocephalus no development was observed. Spirocerca lupi larvae were not detected in the cricket species Gryllus assimilis, or the cockroach species Periplaneta americana, and, similarly, G. ingluvicola larvae were not detected in the millipede species O. kyrhocephalus. The difference in the susceptibility of the arthropods to the two parasite species may depend on their feeding biology.

Spirocerca lupi infection in the dog: a review.

Spirocercosis is a disease occurring predominantly in Canidae, caused by the nematode Spirocerca lupi. Typical clinical signs are regurgitation, vomiting and dyspnoea. The life-cycle involves an intermediate (coprophagous beetle) and a variety of paratenic hosts. Larvae follow a specific migratory route, penetrating the gastric mucosa of the host, migrating along arteries, maturing in the thoracic aorta before eventually moving to the caudal oesophagus. Here the worm lives in nodules and passes larvated eggs which can be detected using zinc sulphate faecal flotation. Histologically, the mature oesophageal nodule is composed mostly of actively dividing fibroblasts. Spirocerca lupi-associated oesophageal sarcomas may occur and damage to the aorta results in aneurysms. A pathognomonic lesion for spirocercosis is spondylitis of the thoracic vertebrae. Primary radiological lesions include an oesophageal mass, usually in the terminal oesophagus, spondylitis, and undulation of the aortic border. Contrast radiography and computed tomography are helpful additional emerging modalities. Oesophageal endoscopy has a greater diagnostic sensitivity than radiography. Endoscopic biopsies are not sensitive for detecting neoplastic transformation. Doramectin is the current drug of choice, effectively killing adult worms and decreasing egg shedding. Early diagnosis of infection is still a challenge and to date no ideal regimen for prophylaxis has been published.

Human thelaziosis--a neglected parasitic disease of the eye.

The oriental eyeworm, Thelazia callipaeda (Spirurida, Thelaziidae), infects a range of definitive hosts, such as dogs, cats, foxes, rabbits, and humans. This parasite usually lives under the nictitating membrane of the eye, where the adult females release first-stage larvae into the lachrymal secretions; these larvae are subsequently ingested by the intermediate arthropod host within which they develop to the infective, third-stage larvae. The latter larvae are then deposited into the eyes of the definitive host. Recently, T. callipaeda has been reported to infect dogs, foxes, and/or cats in Europe (Italy, France, and Germany). Human thelaziosis (HT) is considered to be an underestimated parasitic disease, whose prevalence appears to have increased in poor socioeconomic settings in many Asian countries, including China. In humans, the disease can be subclinical or symptomatic, exhibiting epiphora, conjunctivitis, keratitis, excessive lachrymation, corneal opacity, and/or ulcers. Knowledge about HT is presently fragmentary and mainly limited to clinical case reports. This article provides a background on the parasite and its life cycle, reviews cases of human thelaziosis, summarizes key aspects regarding the diagnosis of thelaziosis, and proposes future research and methods of control of the disease in humans, particularly in Asia.

[An update on the research of human thelaziosis].

Thelaziosis is one of the parasitic zoonoses which affects the eyes of humans and domestic animals. This review covers its distribution, morphology and life cycle of the parasite, pathogenesis, clinical diagnosis, treatment, and prevention of the disease.

EYEWORMS (OXYSPIRURA PETROWI) IN NORTHERN BOBWHITES (COLINUS VIRGINIANUS) FROM THE ROLLING PLAINS ECOREGION OF TEXAS AND OKLAHOMA, 2011-13.

The Northern Bobwhite ( Colinus virginianus ) has been steadily declining throughout much of its historic range for decades. The Rolling Plains ecoregion of Texas and western Oklahoma, historically rich with wild Northern Bobwhites and one of the last remaining quail strongholds, also has a declining population. During August and October in 2011-13, 348 Northern Bobwhites from the Rolling Plains were examined for eyeworms (Oxyspirura petrowi). Of these 348 Northern Bobwhites, 144 (41.4%) were infected with 1,018 total eyeworms. Eyeworm abundance (mean±SE) was 2.9±0.4 (range 0-64), with an intensity (mean±SE) of 7.1±0.6. Eyeworm prevalence was significantly higher in adult Northern Bobwhites (58.7%) than in juveniles (35.4%). Recent research suggests that eyeworms have the potential to cause cellular tissue damage to the eye, but it is unknown how these worms affect host survivability. This study further expands the regional distribution of O. petrowi in Northern Bobwhites in the Rolling Plains ecoregion and assesses the prevalence and abundance of infection across host age, host sex, and year. Further research is warranted on the life history of O. petrowi and assessing the impacts of eyeworms on their definitive host at individual and population levels.

Pathological Response of Northern Bobwhites to Oxyspirura petrowi Infections.

The effects of Oxyspirura petrowi infections in northern bobwhites ( Colinus virginianus ) are not well understood. While studies have reported O. petrowi infections, none has histopathologically examined the eye surface and intraorbital glands to assess cellular-level impacts associated with infection. This study is the first to document the histopathology associated with O. petrowi infections. Oxyspirura petrowi occurred on the eye surface as well as in the conjunctiva, lacrimal ducts, lacrimal glands, and Harderian glands. Histopathology showed infections of O. petrowi caused cellular damage to these tissues, scarring and interstitial keratitis of the cornea, and acinar atrophy of the Harderian gland.

Nematode biology and larval development of Thelazia callipaeda (Spirurida, Thelaziidae) in the drosophilid intermediate host in Europe and China.

Thelazia callipaeda, commonly known as the 'oriental eyeworm', has been recently reported in Italy and other European countries. The insect/s that act as intermediate hosts and details of larval development inside the vector remain unclear. In order to (1) demonstrate the species of fly that may act as vector/s for T. callipaeda in southern Italy (Site A) and China (Site B) and (2) describe the larval development of the nematode in the body of flies, 847 Phortica (Drosophilidae) flies were collected from the above two sites, each with a history of human and/or canine thelaziosis. Flies were identified as Phortica variegata (245 - site A) and Phortica okadai (602 - site B), experimentally infected by 1st-stage larvae (L1), kept at different temperatures and dissected daily until day 180 post-infection (p.i.). Dead flies from site A were subjected to specific polymerase chain reaction (PCR) assay to detect T. callipaeda. To demonstrate the role of Phortica as vectors of T. callipaeda, 3rd-stage larvae (L3) recovered from the proboscis of flies were deposited onto the cornea of the eyes of dogs and rabbits. Following dissection, 3 (2.9%) of P. variegata in site A were found to be infected by L3 in the proboscis on days +14, +21 and +53 p.i., compared with 26 (18.4%) of Phortica flies recorded as being positive by PCR. Sequences from positive PCR products were 99% identical to sequences of the corresponding species available in GenBank (AY207464). At site B, 106 (17.6%) of 602 dissected P. okadai were found to be infected by T. callipaeda larvae (different stages) and in total 62 L3 were recovered from the proboscis of 34 (5.6%) flies. The shortest time in which L3 were found was at day +14, +17, +19, and +50 p.i. respectively, depending on the environmental temperatures. Of 30 flies overwintered for 6 months, 6 L3 were detected at day +180 p.i. in 3 flies (10%). The biology of larval development was reconstructed on the basis of the dissection of 602 P. okadai-infected flies and the morphology of larval stages in the insect body described. The present work provides evidence that P. variegata and P. okadai act as vectors for T. callipaeda in southern Europe and in China, respectively. The phenomenon of overwintering is described here for the first time for T. callipaeda and discussed. Finally, the relationship between T. callipaeda and its fly vector is considered in light of disease prophylaxis and to model its dissemination into habitats and environments favourable to Phortica flies.