First description of a fatal equine infection with Halicephalobus gingivalis in Portugal. Relevance for public health.

Halicephalobus gingivalis is a small saprophytic rhabditid nematode, represented only by females with a typical rhabditoid oesophagus and one egg in the uterus, capable of infecting vertebrates. This opportunistic parasite present in the soil, manure and decaying humus, is thought to penetrate through previous injuries to the mouth, eyes and skin of horses and migrate to various organs. The brain is one such organ, where the females lay their eggs, leading to malacia and causing a sudden onset of neurological signs, such as anorexia, ataxia, urinary incontinence, blindness, decreased menace and tonal reflexes, tremors and aggressiveness. The disease is invariably fatal whenever brain lesions are present, and the diagnosis usually achieved only post-mortem. The present work aims to describe the first case of infection by H. gingivalis ever reported in Portugal. An 8-year old warmblood horse presented with an 8-day history of progressive blindness involving the left eye, initially with normal pupillary reflexes, advancing to bilateral blindness and increasing deterioration in clinical condition. After euthanasia, the animal was submitted for necropsy. Organ samples were collected and fixed in 10% neutral buffered formalin for routine histopathology. A large mass was found in the left kidney corresponding to fibrous tissue heavily infiltrated with inflammatory cells and numerous nematodes. In the brain, multiple, bilateral and asymmetrical foci of malacia containing several rhabditoid nematodes, larvae and zygotes, and high numbers of inflammatory cells were found. The nematodes were identified as H. gingivalis. The clinical history, necropsy and histological findings presented constitute a typical case of H. gingivalis infection in a horse, never previously described in Portugal to the authors' best knowledge. Humans can be infected by contact with contaminated manure, which makes this nematode a public health concern, especially for people living and/or working in close proximity to horses.

Development of Phasmarhabditis hermaphrodita (and members of the Phasmarhabditis genus) as new genetic model nematodes to study the genetic basis of parasitism.

The genetic mechanisms of how free-living nematodes evolved into parasites are unknown. Current genetic model nematodes (e.g. Caenorhabditis elegans) are not well suited to provide the answer, and mammalian parasites are expensive and logistically difficult to maintain. Here we propose the terrestrial gastropod parasite Phasmarhabditis hermaphrodita as a new alternative to study the evolution of parasitism, and outline the methodology of how to keep P. hermaphrodita in the lab for genetic experiments. We show that P. hermaphrodita (and several other Phasmarhabditis species) are easy to isolate and identify from slugs and snails from around the UK. We outline how to make isogenic lines using 'semi-natural' conditions to reduce in-lab evolution, and how to optimize growth using nematode growth media (NGM) agar and naturally isolated bacteria. We show that P. hermaphrodita is amenable to forward genetics and that unc and sma mutants can be generated using formaldehyde mutagenesis. We also detail the procedures needed to carry out genetic crosses. Furthermore, we show natural variation within our Phasmarhabditis collection, with isolates displaying differences in survival when exposed to high temperatures and pH, which facilitates micro and macro evolutionary studies. In summary, we believe that this genetically amenable parasite that shares many attributes with C. elegans as well as being in Clade 5, which contains many animal, plant and arthropod parasites, could be an excellent model to understand the genetic basis of parasitism in the Nematoda.

Histologic characterization of eosinophilic encephalitis in horses in Florida.

Eosinophils within the central nervous system are abnormal and are usually associated with fungal or parasitic infections in horses. Causative agents include Halicephalobus gingivalis, Sarcocystis neurona, and Neospora hughesi. Confirmation of these organisms via specific testing is typically not performed, and final diagnoses are often presumptive. With molecular technology, many of these organisms can now be confirmed. This is important for emerging and zoonotic pathogens, including Angiostrongylus cantonensis, an emerging parasite of interest in the southeastern United States. We retrospectively analyzed eosinophilic encephalitides in Floridian horses for H. gingivalis, S. neurona, and A. cantonensis, applied descriptors to equine eosinophilic encephalitides, and determined if a relationship existed between these descriptions and specific etiologic agents. In a database search for horses with eosinophilic and other encephalitides submitted to the University of Florida, College of Veterinary Medicine, Anatomic Pathology Service, we identified 27 horses with encephalitis, and performed DNA isolation and rtPCR on formalin-fixed, paraffin-embedded blocks from these cases. Real-time PCR identified 6 horses positive for S. neurona and 4 horses positive for H. gingivalis; all horses were negative for A. cantonensis. All 25 control horses were negative for H. gingivalis, S. neurona, and A. cantonensis. Pattern analysis and eosinophil enumeration were not useful in differentiating among causes of eosinophilic encephalitides in horses in our study.

Isolation and molecular characterisation of Halicephalobus gingivalis in the brain of a horse in Piedmont, Italy.

BACKGROUND: A fatal case of meningoencephalitis was reported in a 13-year-old Koninklijk Warmbloed Paard Nederland stallion, suspected of West Nile virus (WNV) infection, in the Piedmont region of Italy. Clinical signs included right head tilt and circling, depression alternated with excitability, fever and lateral strabismus. Combined treatment consisting of dimethylsulfoxide, dexamethasone, sulphonamides and sedative was administered, but because of the poor conditions the horse was euthanatized and submitted for necropsy. RESULTS: At post-mortem examination no skin lesions were observed, all organs appeared normal on gross evaluation and only head and blood samples were further investigated. Neuropathological findings consisted of granulomatous meningoencephalitis and larvae and adult females of Halicephalobus gingivalis were isolated and identified from the digested brain. Frozen brain was submitted to PCR amplification and 220 bp multiple sequence alignment was analysed by Bayesian phylogenetic analysis. CONCLUSIONS: Phylogenetic inference revealed that the isolate belongs to H. gingivalis Lineage 3. WN surveillance can help to deepen our knowledge of horse neurological disorders investigating their causes and incidence. Moreover, it can help to understand the geographic distribution of the H. gingivalis, to unravel epidemiological information, and to estimate risk for humans.

Rhinella marina (Amphibia: Bufonidae) Versus Rhabdias paraensis (Nematoda: Rhabdiasidae): Expanding the View on a Natural Infection.

Amphibian and reptile lungs are frequently infected with Rhabdias parasites, and this condition ultimately leads to reduced survival, performance, and growth because of granulomatous inflammation, nodule formation, and nematodal pneumonia onset. Here we investigate the histopathological features of naturally infected Rhinella marina by the lung nematode Rhabdias paraensis. A total of 10 host animals were captured in peridomiciliar areas in the eastern Brazilian Amazon, and anatomic-histological analyses were performed on both the infected and non-infected lungs of these amphibians. Helminths were usually found within the secondary and primary septa of infected lungs whereas parasites were not detected within vessels or adhering to tissues. In addition, we observed discrete erythrocytes, diapedesis foci, few granulocytes and erythrocytes in the interseptal spaces, discrete cell infiltration, and a small number of melanomacrophages, and no granulomas or cysts were observed. New aspects related to changes in tissue and helminth-host interactions are discussed for the relationship of R. paraensis × Rhi. marina from the Amazon region.

An outbreak of bovine meningoencephalomyelitis with identification of Halicephalobus gingivalis.

Halicephalobus gingivalis is an opportunistic parasite which is known to cause fatal meningoencephalomyelitis primarily in equines but sporadically also in humans. In April 2014, laboratory examination of the head of a young dairy calf, euthanized due to severe central nervous system symptoms, revealed the presence of granulomatous to necrotizing encephalitis and myriads of nematodes in the brain lesion. Morphologically the parasites were identified as H. gingivalis. The diagnosis was confirmed by molecular analysis of the large subunit (LSU) rRNA and the small subunit (SSU) rRNA genes, revealing genetic variations of 0.5-4.4% and 0.7-8.6%, respectively, between the H. gingivalis isolated from the Danish calf and published isolates, collected worldwide from free-living and parasitic stages of the nematode. Clinical symptoms and histological changes indicated infection with H. gingivalis from another three calves in the herd. This is the first scientific publication of H. gingivalis induced meningoencephalomyelitis in ruminants. As ante mortem diagnosis is a major challenge, the infection may easily remain undiagnosed in cattle.

First report of fatal systemic Halicephalobus gingivalis infection in two Lipizzaner horses from Romania: clinical, pathological, and molecular characterization.

Halicephalobus gingivalis (H. gingivalis) causes a rare and fatal infection in horses and humans. Despite the zoonotic potential and severity of the disease, the epidemiology and pathogenesis of halicephalobiasis are still poorly understood. Several European cases of equine halicephalobiasis have been documented; however, in South-Eastern European countries, including Romania, equine neurohelminthiasis caused by H. gingivalis has not been previously described. Two Lipizzaner horses with a clinical history of progressive neurological signs were referred to the Pathology Department of the Cluj-Napoca (Romania) for necropsy. Both horses died with severe neurological signs. Gross examination and cytological, histological, and molecular analyses were performed. The stallions came from two different breeding farms. No history of traveling outside Romania was recorded. At necropsy, granulomatous and necrotizing lesions were observed in the kidneys, lymph nodes, brain, retroperitoneal adipose tissue, and lungs, indicating a systemic infection. Parasitological and histopathological analyses evidenced larval and adult forms of rhabditiform nematodes consistent with Halicephalobus species. Parasites were observed in both lymph and blood vessels of different organs and were also identified in urine samples. A subunit of the large-subunit ribosomal RNA gene (LSU rDNA) of H. gingivalis (673 bp) was amplified from lesions in both horses.To the authors' knowledge, this is the first report of equine systemic H. gingivalis infection in Romania and in South-Eastern Europe. Our findings provide new insights into the geographic distribution of specific genetic lineages of H. gingivalis, while also raising public health awareness, as the parasite is zoonotic.

Subspectacular nematodiasis caused by a novel Serpentirhabdias species in ball pythons (Python regius).

Subspectacular nematodiasis was diagnosed in three captive-bred juvenile ball pythons (Python regius) from two unrelated facilities within a 6-month period. The snakes were presented with similar lesions, including swelling of facial, periocular and oral tissues. Bilaterally, the subspectacular spaces were distended and filled with an opaque fluid, which contained nematodes and eggs. Histopathology showed nematodes throughout the periocular tissue, subspectacular space and subcutaneous tissue of the head. The nematodes from both facilities were morphologically indistinguishable and most closely resembled Serpentirhabdias species. Morphological characterization and genetic sequencing indicate this is a previously undescribed rhabdiasid nematode.

First human case of fatal Halicephalobus gingivalis meningoencephalitis in Australia.

Halicephalobus gingivalis (previously Micronema deletrix) is a free-living nematode known to cause opportunistic infections, mainly in horses. Human infections are very rare, but all cases described to date involved fatal meningoencephalitis. Here we report the first case of H. gingivalis infection in an Australian human patient, confirmed by nematode morphology and sequencing of ribosomal DNA. The implications of this case are discussed, particularly, the need to evaluate real-time PCR as a diagnostic tool.

Meningoencephalitis caused by Halicephalobus gingivalis in a thoroughbred gelding.

A 6-year-old Thoroughbred gelding was euthanized after a 2-month period of abnormal neurological signs, such as circling left in his pen and hitting his head and body against the wall. After the horse was euthanized on the farm, a half of the brain and whole blood were submitted for diagnostic tests. Histopathological examination of the brain revealed granulomatous and eosinophilic meningoencephalitis with numerous intralesional nematodes, predominantly affecting the cerebrum. Multifocal malacic foci were scattered in the brain parenchyma. The intralesional parasites were identified as Halicephalobus gingivalis by morphological features and PCR testing. To the best of our knowledge, this is the first report of meningoencephalitis caused by H. gingivalis in the horse in Korea.

Halicephalobus gingivalis: a rare cause of fatal meningoencephalomyelitis in humans.

The genus Halicephalobus consists of eight species of free-living nematodes. Only one species (H. gingivalis) has been reported to infect vertebrates. Human infection is extremely rare, and only four cases have been reported in the literature. These nematodes seem to exhibit neurotropism, but their life cycle, mode of infection, and risk factors are poorly understood. Neurohelminthiases are not commonly recognized in the United States and when they do occur, pose great diagnostic challenges because of lack of appropriate non-invasive screening and/or confirmatory tests. We report a challenging case of meningoencephalomyelitis caused by a Halicephalobus sp., in which the patient had a rapidly deteriorating clinical course. The case did not raise any clinical suspicion of neurohelminthiases, although increased eosinophils were present in the cerebrospinal fluid. This case presents an opportunity to highlight the importance of considering parasitic infection in meningoencephalitis or meningoencephalomyelitis presenting atypically.

Pulmonary lesions caused by the nematode Rhabdias fuscovenosa in a grass snake, Natrix natrix.

In June 2005, a free-ranging grass snake, Natrix natrix infected with Rhabdias fuscovenosa was collected for histologic examination of the lungs. Gross lesions were not noted; however, histologic examination revealed vacuolar degeneration of the respiratory epithelium, hemorrhage, smooth muscle degeneration, faveolae necrosis and obstruction, and intralesional nematodes. Eosinophils were the most common inflammatory cell observed in the areas of necrosis and interstitium. Herein we describe histopathologic changes due to R. fuscovenosa in a free-ranging grass snake.

Sarcoptic mange and Pelodera dermatitis in an American black bear (Ursus americanus).

An adult female free-ranging American black bear (Ursus americanus) was presented in poor body condition, with advanced skin disease. Skin changes included hair loss, lichenification, crusting, and focal erosions. Skin scrapings and histopathology identified two distinct parasitic conditions that were contributing to this animal's dermatitis. Large numbers of larvae, nymphs, and adults of Sarcoptes scabiei were present in the superficial epidermis, and nematodes consistent with Pelodera strongyloides were abundant within the hair follicles. This appears to be the first reported case of Pelodera dermatitis in a bear species, adding a new differential agent to the list for dermatitis in bears. The sarcoptic mange and poor body condition of this bear may have been contributing factors to the development of the opportunistic Pelodera infestation.

Lungworm infection in three loggerhead sea turtles, Caretta caretta.

Although lungworms are known to infect many mammalian species and a few are known to infect snakes, lizards, and birds, previously none were known to infect chelonians. This study documents the first three known cases of lungworms in loggerhead sea turtles, Caretta caretta. It is unlikely that the lungworms were the primary cause of illness in any of the cases, and they may be only contributory or even incidental. Changes observed in the two cases that died included tracheal and bronchial epithelial hyperplasia and goblet cell hyperplasia. Lesions caused directly by the parasites seem to be restricted to the upper respiratory tree (trachea and main bronchi), but changes in the lungs themselves may be caused by the debris produced by the worms. Although neither case was successfully treated for the lungworms prior to death, it would appear that oxfendazole may be an effective treatment and has been used in the third case that is still undergoing rehabilitation.

Halicephalobus gingivalis-associated meningoencephalitis in a Thoroughbred foal.

A 13-week-old Thoroughbred colt from central Kentucky was euthanized after an acute onset of ataxia, blindness, head tremors, leaning to the right, recumbency, and seizures. Microscopically, there was a verminous meningoencephalitis characterized by an eosinophilic and granulomatous inflammatory reaction primarily affecting the cerebellum. Dispersed within regions of inflammation were numerous cross and longitudinal sections of intact and degenerative small nematodes. The nematodes had dorsoflexed ovaries and ventroflexed vulvas, which are distinguishing features of Halicephalobus gingivalis. Intact nematodes, compatible with H. gingivalis, also were recovered and identified from portions of the brain that had been frozen for 5-week post-necropsy examination via tissue maceration and additional laboratory techniques.

Pelodera (syn. Rhabditis) strongyloides as a cause of dermatitis--a report of 11 dogs from Finland.

BACKGROUND: Pelodera (Rhabditis) strongyloides is a small saprophytic nematode that lives in decaying organic matter. On rare occasions, it can invade the mammalian skin, causing a pruritic, erythematous, alopecic and crusting dermatitis on skin sites that come into contact with the ground. Diagnosis of the disease is based on case history (a dog living outdoors on damp straw bedding) with characteristic skin lesions and on the demonstration of typical larvae in skin scrapings or biopsy. Pelodera (rhabditic) dermatitis cases have been reported mainly from Central European countries and the United States. CASE PRESENTATION: During 1975-1999, we verified 11 canine cases of Pelodera dermatitis in Finland. The cases were confirmed by identifying Pelodera larvae in scrapings. Biopsies for histopathology were obtained from three cases, and typical histopathological lesions (epidermal hyperplasia, epidermal and follicular hyperkeratosis, folliculitis and furunculosis with large numbers of nematode larvae of 25-40 microm of diameter within hair follicles) were present. The Pelodera strongyloides dermatitica strain from the first verified case in Finland has been maintained in ordinary blood agar in our laboratory since 1975. Light microscopy (LM) and scanning electron microscopy (SEM) studies were employed to obtain detailed morphological information about the causative agent. The rhabditiform oesophagus at all developmental stages, the morphology of the anterior end of the nematode, copulatory bursa and spicules of the male and the tail of the female were the most important morphological features for identifying P. strongyloides. CONCLUSION: These cases show that Pelodera dermatitis occurs in Finland, and also farther north than described earlier in the literature. This condition should be considered when a dog living outdoors has typical skin lesions situated at sites in contact with the ground as the main presenting clinical feature. The fastest and easiest way to confirm the diagnosis is to demonstrate typical larvae in skin scrapings. In uncertain cases, skin biopsy and culturing of the worms are recommended as supplementary diagnostic procedures.

[Halicephalobus gingivalis infection in a 5-year-old Tinker gelding]. / Halicephalobus gingivalis infectie bij een vijfjarige Tinker ruin.

A 5-year old Tinker gelding was referred to the Department of Equine Sciences with a left eye uveitis and fever. At presentation the horse showed a mild lethargy, fever and decreased vision of the left eye. Rectal examination revealed an enlarged left kidney, with a hard and an irregular surface. The cranial mesentery artery had an enlarged and irregular aspect. Blood analysis showed anaemia, leucocytosis, increased blood urea nitrogen and creatinine and a hyperproteinemia. Urine analysis repeatedly showed a marked proteinuria and an increased gammaGT/creatinine ratio. The amount of abdominal fluid was slightly increased. However, the aspect, amount of cells and protein were normal. In the following two days the fever persisted and the horse showed anorexia and severe neurological signs. The horse was euthanized with permission of the owner. Post mortem examination showed a generalized parasitic infestation of Halicephalobus gingivalis in the uvea of the left eye, the kidneys and the central nerve system.

Halicephalobus gingivalis (Nematoda) infection in a Grevy's zebra (Equus grevyi).

A 6-yr-old female Grevy's zebra (Equus grevyi) with a disseminated rhabditiform nematode infection is described. Antemortem clinical signs were limited to blindness and abnormal behavior believed to be caused by a recurrent nematode-induced uveitis. Histologic examination of the kidneys, heart, eyes, uterus, and lymph nodes revealed granulomas containing multiple sections of rhabditiform nematodes. Most of the recovered nematodes were larval stages with only a few adult females noted. The adults measured 243-297 microm x 11-16 microm (x = 269 x 14 microm). The distinctive rhabditiform esophagi had corpus:isthmus:bulb proportions of 19:11:5. On the basis of adult morphology, the nematode was identified as Halicephalobus gingivalis. This is the first report of this parasite in a zebra and indicates that this parasitic granulomatous disease should be considered in zebras with neurologic disease.