Parasitology Research

, Volume 115, Issue 3, pp 1097–1103

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

  • Marian A. Taulescu
  • Angela M. Ionicã
  • Eva Diugan
  • Alexandra Pavaloiu
  • Roxana Cora
  • Irina Amorim
  • Cornel Catoi
  • Paola Roccabianca
Original Paper

DOI: 10.1007/s00436-015-4839-7

Cite this article as:
Taulescu, M.A., Ionicã, A.M., Diugan, E. et al. Parasitol Res (2016) 115: 1097. doi:10.1007/s00436-015-4839-7

Abstract

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.

Keywords

Equine Halicephalobus gingivalis Granulomatous nephritis Systemic infection Fatal disease 

Introduction

Halicephalobiasis represents a systemic infection caused by helminths of the genus Halicephalobus. The genus Halicephalobus in the Order Rhabditida, Family Panagrolaimidae, consists of nine species: H. gingivalis, H. limuli, H. similigaster, H. minutum, H. parvum, H. palmaris, H. intermedia, H. laticauda, and H. brevicauda (Andrássy, 1984). Of these, only H. gingivalis (syn. Micronema deletrix) has been reported to infect vertebrates and was described for the first time by Stefanski in 1954 as a free-living, saprophytic nematode belonging to the Order Tylenchida (Class Chromadorea).

These species are commonly found in soil and decaying humus (Nadler et al., 2003), but Halicephalobus gingivalis nematodes have been sporadically associated with systemic infection in horses (Akagami et al. 2007; Hermosilla et al. 2011; Eydal et al. 2012; Henneke et al. 2014; Jung et al. 2014), in one donkey (Schmitz and Chaffin, 2004), in one Grevy’s zebra (Equus grevyi) (Isaza et al. 2000), and in a Black Angus cow (Montgomery and O’Toole, 2006). Several cases have also been reported in humans (Anwar et al. 2015; Lim et al. 2015; Monoranu et al. 2015).

The majority of H. gingivalis infections in horses have been fatal and are usually diagnosed after death and, very rarely, ante-mortem (Henneke et al. 2014). Neurological signs, including ataxia, have been reported in several horses and rapidly evolve into a fatal disease (Bryant et al. 2006).

In the present report, we illustrate the first two cases of equine systemic H. gingivalis infection diagnosed in South-Eastern Europe, specifically in Romania. Clinical, pathological, and molecular features of the cases and differential diagnosis are discussed.

Materials and methods

Clinical history

Two Lipizzaner horses used for breeding in two different stud farms from Transylvania (the central region of Romania), with no previous history of medical problems, are included. Horses had not traveled outside Romania.

The first case (horse A) was a 14-year-old black Lipizzaner stallion (Siglavy Capriola XXIII). The stallion developed sudden depression, anorexia, and reluctance to move. Physical examination showed normal temperature, breathing, heart rate, and peristalsis. Approximately 8 h from initial signs, the animal began circling and developed neurological visual impairment (differentiated only between light and dark), with normal pupillary light reflex. The horse was able to avoid the door post only when the box was open and light came through the opening, but hit the door post under reduced light conditions (door closed). The stallion had the first seizure 12 h after the first signs. For the next 2 days, its condition worsened and seizures developed at shorter intervals. During seizures, the stallion had bruxism and muscular contractures of the neck and developed self-mutilating behavior by hitting the walls and posts of the box (when not sedated). The stallion died in May 2014, after a week of hypothermic episodes and progressive worsening of neurological signs.

The second case (horse B) was a 10-year-old Lipizzaner stallion (Siglavy Capriola XXVII). The stallion suddenly developed a wide base stance of the posterior legs, astasia-abasia, followed by ataxia. In the following hours, the stallion developed muscle contractions and fasciculations of the left gluteal region with pain and swelling on palpation. Mucous membranes were anemic. The core temperature was 37.4 °C, the pulse was irregular (58 beats per minute), with 15 breaths per minute and a shallow breathing pattern. The animal’s clinical signs progressed, but the appetite was preserved. The stallion was treated symptomatically, to no avail, and later in the day, the animal presented hyperkinesia, with clonic contractions both in the hind and front limbs, muscle fasciculations, and heat and swelling of the limbs. Cranial nerve examination revealed mydriasis, nystagmus, bilateral cortical visual impairment that progressed to central blindness, bruxism, and opisthotonus (increased extensor tone of the limbs and the neck) alternating with pleurosthotonus (lateral flexion of the whole body). Due to progressive muscle weakness, the animal adopted decubitus and could only stand if assisted.

Medications included intravenous fluids, vitamins, minerals, nonsteroidal anti-inflammatory drugs, sodium bicarbonate, sedation (Xylazine-Xylazin Bio 2 %, Bioveta, Detomidine-Domosedan, Pfizer Animal Health) and antimicrobials (Metronidazole, Cefquinome-Cobactan, MSD Animal Health). After 2 days, the stallion adopted lateral recumbency, started pedaling, became anorexic and adipsic, and became comatose. The stallion died of cardiopulmonary arrest 3 days later. Anamnesis revealed that two half brothers had previously died presenting similar clinical signs.

Differential diagnoses for both cases included metabolic/toxic and inflammatory conditions such as meningitis (bacterial, fungal, or viral etiologies).

Both horses were submitted for necropsy to the Pathology Department of the Faculty of Veterinary Medicine from Cluj-Napoca (Romania).

Postmortem examination

Necropsies were performed about 6 h (horse A) and 12 h (horse B) after death, respectively.

Samples from the kidneys, lumbar and inguinal lymph nodes, retroperitoneal adipose tissue, lungs, central nervous system (cerebrum, midbrain, cerebellum, medulla oblongata, pons, and spinal cord), heart, skin, eyes, lips, tongue, stomach, liver, urinary bladder, and skeletal muscle tissue were collected for cytological, histological, and molecular analyses.

For cytological analysis, imprints from the kidneys, right lumbar lymph nodes, and brain were stained by Dia Quick-Panoptic (DQP, Reagent Ltd., Budapest, Hungary).

For histological examination, samples were fixed in 10 % phosphate-buffered formalin (pH 7.0) for 24 h, routinely processed, embedded in paraffin wax, cut into 3–4-μm sections, and stained with hematoxylin and eosin (H & E).

For parasitology, nematodes were extracted from frozen renal tissues and lymph nodes for identification. Parasites were suspended in sterile saline (NaCl) 0.9 % solution and examined under glass coverslips without glycerin as previously described (Akagami et al. 2007). About 50 ml of urine was collected from both horses in a sterile plastic container in order to evaluate for the presence of parasites.

Samples were examined using an Olympus BX51 microscope. Photomicrographs and measurements of the parasites for morphologic identification were taken using an Olympus SP 350 digital camera and Cell^B basic imaging software (Olympus Corporation, Japan).

DNA extraction and molecular identification

Genomic DNA was extracted from lesional tissues (kidneys and lumbar lymph nodes in horse A; kidneys and brain in horse B) using a commercial kit (Isolate II Genomic DNA Kit, Bioline, UK) according to the manufacturer’s instructions.

A region of the nuclear large subunit ribosomal RNA gene (LSU rDNA) of H. gingivalis (673 bp) was amplified using 632/634 primer pair, as previously described (Nadler et al. 2003). PCR products were visualized by electrophoresis in a 2 % agarose gel stained with RedSafeTM 20,000× Nucleic Acid Staining Solution (Chembio, UK), and their molecular weight was assessed by comparison to a molecular marker (O’GeneRulerTM 100 bp DNA Ladder, Thermo Fisher Scientific Inc., USA).

PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN) and then analyzed by sequence analysis (performed at Macrogen Europe, Amsterdam). Sequences were compared to those available in GenBank™ dataset by Basic Local Alignment Tool (BLAST) analysis.

Results

At necropsy, severe dehydration and several decubitus ulcers over the head, thorax, legs, and lumbar area were detected. Ulcers were more severe in horse A and were associated with numerous 3–5 mm subacute, irregular erosions and ulcers on the inner side of the inferior lip, interpreted as self-trauma.

Kidneys were markedly enlarged and deformed by numerous bulging, grayish-white nodules, varying from 1–4 cm (horse B) to 5–10 cm (horse A) in diameter (Figs. 1 and 2). In both cases, the renal, lumbar, and inguinal lymph nodes were diffusely enlarged up to 2–3 times the normal size, irregular in shape, and white to gray on cross section. The brain was characterized by hyperemia and edema, and few random, small, and red foci were observed on the cut sections of the cortex. The lungs failed to collapse and displayed severe hyperemia and edema. In horse A, several gastric erosions and subacute ulcers were identified in the glandular mucosa. No other significant gross lesions were evidenced in other organs.
Figs. 1–8

1 Kidneys of horse A characterized by multiple white to gray pyogranulomas. 2 Longitudinal section of the kidney (horse A) showing corticomedullary gray-tan firm masses in association with H. gingivalis infection. 3 Kidney, horse B. Diffuse, locally extensive, chronic granulomatous nephritis with severe atrophy of the adjacent renal parenchyma (interrupted line). Hematoxylin and eosin stain. Bar = 100 μm. 4 Kidney, horse B. Granulomatous inflammation composed of numerous multinucleated giant cells (red arrow) centered on longitudinal and cross sections of nematodes consistent with H. gingivalis (black arrow). Hematoxylin and eosin stain. Bar = 50 μm. 5 Kidney, horse B. Longitudinal section of H. gingivalis within the granulomatous reaction. Note the characteristic rhabditiform esophagus (arrowhead). Hematoxylin and eosin stain. Bar = 10 μm. 6 Kidney, horse B. The renal tubules are dilated and filled with foamy macrophages (red arrow) and numerous fragments of nematodes (black arrow). Hematoxylin and eosin stain. Bar = 20 μm. 7 Direct examination of centrifuged urine from horse B demonstrating numerous nematodes consistent with H. gingivalis admixed with calcium carbonate crystals. Bar = 100 μm. 8 Direct examination of dissected frozen renal tissue from horse A containing an adult nematode with rhabditiform esophagus and tapered, pointed tail and 20/15 μm in diameter, unembryonated (8a) and embryonated eggs (8b) (Bar = 10 μm); (Bar = 100 μm) (Fig. 8)

Microscopically, the kidney were severely effaced and replaced by numerous, well-delimited, variably sized, and occasionally confluent pyogranulomatous nodules that compressed the renal parenchyma from the capsule to the medulla (Fig. 3). Pyogranulomas showed a thin peripheral concentric fibrous capsule infiltrated by large numbers of lymphocytes and plasma cells and few eosinophils that surround sheets of numerous epithelioid macrophages, numerous multinucleated giant cells (MGCs) containing up to 10–15 haphazardly arranged nuclei (foreign body type) or the nuclei were arranged in a horseshoe fashion (Langhans type), and a central core filled with large numbers of neutrophils and eosinophils. Many granulomas contained multiple tangential and cross sections of well-preserved larval and adult female nematodes (Fig. 4). Adult nematodes were 15 to 25 μm in diameter and up to 200 μm in length, with a smooth cuticle, platymyarian-meromyarian musculature, a long rhabditiform esophagus composed of a corpus, isthmus, and bulb, numerous deeply basophilic 2–4-μm internal structures, tubular digestive tract, tapered tail, and a pseudocoelom which were often visible. The nematodes were identified as Halicephalobus spp. (Fig. 5). Multifocally, the renal parenchyma was completely replaced by fibrous tissue associated with mild infiltrates of macrophages, lymphocytes, and lesser neutrophils. Multifocally, the collecting renal tubules were severely distended, lined by an attenuated epithelium, and filled with numerous fragments of nematodes admixed with large numbers of foamy macrophages and scattered neutrophils (Fig. 6). In some areas, the glomeruli were atrophied with severe fibrosis of Bowman’s membrane. By direct examination of centrifuged urine, numerous nematodes with the same morphology admixed with calcium carbonate crystals were identified (Fig. 7). Adults, larvae with rhabditiform esophagus and tapered pointed tail, 20/15 μm in diameter, and oval unembryonated and embryonated eggs were obtained by direct examination of renal samples (Fig. 8). Histopathological findings within the brain were consistent with a verminous meningoencephalitis, predominantly affecting the cortex, beneath the meningeal tissue and around the lateral ventricles. Diffuse congestion, small, well-delimited and random necrotic foci were identified within the midbrain, occipital and parietal lobes of the cerebral cortex, and thalamus. In the malacic areas, numerous Gitter cells, mature lymphocytes, axonal swelling, and spongiosis associated with few parasitic organisms were observed (Figs. 9 and 10). Within the meninges, mild inflammation composed of macrophages, lymphocytes, eosinophils, and scattered neutrophils was observed; numerous nematodes, including larvae and adults, were also present within the CNS affected areas (Fig. 9). No lesions were identified in the spinal cord.
Figs. 9–14

9 Photomicrograph of brain tissue from horse A containing H. gingivalis (arrows) and minimal degeneration and necrosis of the nervous tissue. Hematoxylin and eosin stain. Bar = 50 μm. Inset (imprint smear of brain): nematode with rhabditoid esophagus surrounded by Gitter cells. DQP stain. Bar = 20 μm. 10 Photomicrograph of the brain from horse A with diffuse hyperemia, degeneration, and necrosis of neurons and neuropil (delimited area) and a mild infiltration with foamy macrophages (Gitter cells), lymphocytes, and neutrophils. Hematoxylin and eosin stain. Bar = 20 μm. 11 Photomicrograph of lumbar lymph node from horse A revealing parasites admixed with macrophages, lymphocytes, and neutrophils within the subscapular lymphatic sinus (arrow). Hematoxylin and eosin stain. Bar = 20 μm. Inset, blood vessel partially occluded by a hyaline thrombus enclosing leukocytes and nematodes (arrow). Hematoxylin and eosin stain. Bar = 20 μm. 12 Retroperitoneal fat, horse A. The normal architecture of the adipose tissue is multifocally substituted by a granulomatous reaction centered on parasitic organisms consistent with H. gingivalis (arrow). Hematoxylin and eosin stain. Bar = 50 μm. 13 Photomicrograph of a lung from horse A showing diffuse hyperemia and alveolar edema, and a well-delimited focus composed of parasitic organisms admixed with histiocytes and lymphocytes. Hematoxylin and eosin stain. Bar = 50 μm. 14 Electrophoretic profile: M = molecular marker, lane 1 = sample extracted from kidney, lane 2 = sample extracted from lymph node; horse A

Lymph nodes were severely and diffusely infiltrated, and the subscapular sinuses were markedly distended by macrophages, lymphocytes, and neutrophils. Lymphatic sinuses contained large numbers of parasites (Fig. 11). Multifocally, capillaries were partially occluded by hyaline thrombi embedding leukocytes and nematodes (Fig. 11). Similar lesions were observed in the retroperitoneal adipose tissue (Fig. 12) and pulmonary parenchyma (Fig. 13) in both horses.

Lesions from the stomach and lips did not contain parasites. No additional microscopic lesions were evidenced in the other examined organs.

H. gingivalis LSU rDNA was amplified in both cases (Fig. 14). BLAST analysis of attained sequences showed a 100 % homology to a Japanese strain of H. gingivalis deposited in GenBank (accession number AB289345) and were 99 % similar to other isolates originating from the USA (accession number AY294179) and Korea (accession number KF662357).

Discussion

Free-living environmental nematodes which frequently may infect horses, particularly while grazing, include strongyles, Strongyloides spp., and Halicephalobus spp. (Nadler et al. 2003; Kuzmina, 2012; Lyons and Tolliver, 2014). H. gingivalis causes a fatal infection in animals and humans. At present, various cases of this infection have been reported, but the epidemiology and pathogenesis of this disease are still poorly understood (Kinde et al. 2000).

Equine halicephalobiasis has been reported in several European countries including Denmark (Henneke et al. 2014), UK (Hermosilla et al. 2011), Belgium (Fonderie et al. 2012), and Italy (Di Francesco et al. 2012) and non-European countries including Japan (Akagami et al. 2007) and the USA (Adedeji et al. 2015). In humans, meningoencephalitis caused by H. gingivalis has been described in Australia (Lim et al. 2015), USA (Anwar et al. 2015), and Germany (Monoranu et al. 2015). Despite the wide distribution range of H. gingivalis, the two cases herein described represent the first record of equine halicephalobiasis in Romania and, to the best of our knowledge, in South-Eastern Europe. The autochthonous nature of H. gingivalis in Romania is supported by the history of the two horses, which had never traveled outside the country. Furthermore, the case of horse B is highly suggestive for local endemicity of the parasite, given that the half brothers from the same farm had previously died showing similar clinical signs.

Several aspects regarding the life cycle, infection routes, and tissue migration of the parasites are still under debate. Penetration of mucosal membranes and/or skin is the most common infection routes described (Pearce et al. 2001; Muller et al. 2008). In a recent report of H. gingivalis infection in two foals, a possible prenatal, perinatal, or transmammary transmission was hypothesized (Spalding et al. 1990). After penetrating the host barrier, nematodes can either become encircled by local inflammation or may enter the bloodstream/lymph and disseminate throughout the body (Bröjer et al. 2000). Similar to previous reports (Akagami et al. 2007; Henneke et al. 2014), in these two Lipizzaners, parasites were observed in both lymph and blood vessels. Additionally, there was no evidence of trauma or wounds prior to the development of clinical signs; thus, the way of transmission remains uncertain for both cases. Even if the transmission of H. gingivalis between animals by direct contact has not been ruled out, the two horses described in these reports were never in close contact, indicating the existence of two separate foci.

H. gingivalis infection in horses is generally diagnosed after death and, only very rarely, ante-mortem (Ferguson et al. 2008). However, for the presence of larvae in the urine samples, demonstrated in a previously published (Kinde et al. 2000) and in the current study, the examination of urine could be recommended as an intra-vitam clinical diagnostic method.

H. gingivalis mainly causes necrotizing and pyogranulomatous inflammation in the central nervous system (Henneke et al. 2014) and kidneys (Kinde et al. 2000) in horses. However, its localization in other organs, including the maxillary bone (Fonderie et al. 2012), eyes, and optic nerves, have also been recently reported (Kinde et al. 2000). In single case reports, the nematodes have also been found microscopically in the heart, blood vessels, testicles, femur, nasal bones, preputium, urine, semen, and cerebrospinal fluid (Spalding et al. 1990; Kinde et al. 2000; Muller et al. 2008; Adedeji et al. 2015). The distribution of lesions and parasites in the two cases reported herein can be considered typical for the systemic disease, as they were detected in the kidneys, lymph nodes, brain, lungs, and retroperitoneal adipose tissue.

In horses, clinical signs associated with H. gingivalis infection are various, nonspecific, and related to the organ or system/s affected (Jung et al. 2014). In the present report, the main clinical complaint was the severe neurological condition; however, the manifestations still remained nonspecific. Causes of ataxia in horses include trauma, degenerative myelopathy, Wobbler’s syndrome, neoplasia, Sarcocystis neurona infection, and other infectious agents (Dame et al. 2000; Kinde et al. 2000). In fact, numerous parasitic infections are associated with lesions of the CNS in horses and include Hypoderma lineatum, Hypoderma bovis, Strongylus vulgaris, Draschia megastoma, Setaria sp., and hydatid cysts of Echinococcus granulosus (George, 1990).

Even if H. gingivalis is morphologically distinct from the other species (Anderson et al. 1998), a specific diagnosis is not possible on the sole basis of histopathology. For the definitive diagnosis and the distinction from other parasites, isolation and molecular diagnostic methods, such as PCR, are necessary (George, 1990; Papadi et al. 2013).

Previous studies have shown that genetically homogenous isolates of H. gingivalis have a wide geographical distribution (Nadler et al. 2003). Based on LSU rDNA, the Romanian isolates showed a 100 % homology to a Japanese strain of H. gingivalis and were 99 % similar to other isolates originating from the USA (accession number AY294179) and Korea (accession number KF662357). This finding provides novel information of the geographic distribution of the genetic lineages of H. gingivalis.

Conclusion

This study contributes to a better understanding of the pathogenesis and the epidemiology of equine halicephalobiasis, providing new insights into the geographical distribution of the parasite and of the disease. Our findings confirm that H. gingivalis infection represents a severe systemic and fatal disease that needs to be included in the list of possible differential diagnoses in equines developing sudden nervous clinical signs also in Southern Europe. Additionally, our findings warrant caution for operators working in equine Romanian rearing facilities due to the zoonotic potential of the disease.

Acknowledgments

This paper was published under the frame of European Social Fund, Human Resources Development Operational Program 2007–2013, project no. POSDRU/159/1.5/S/136893.

Compliance with ethical standards

Competing interests

The authors declare that they have no competing interests.

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Marian A. Taulescu
    • 1
  • Angela M. Ionicã
    • 2
  • Eva Diugan
    • 3
  • Alexandra Pavaloiu
    • 4
  • Roxana Cora
    • 1
  • Irina Amorim
    • 5
    • 6
    • 7
  • Cornel Catoi
    • 1
  • Paola Roccabianca
    • 8
  1. 1.Department of Pathology, Faculty of Veterinary MedicineUniversity of Agricultural Sciences and Veterinary MedicineCluj-NapocaRomania
  2. 2.Department of Parasitology and Parasitic Diseases, Faculty of Veterinary MedicineUniversity of Agricultural Sciences and Veterinary MedicineCluj-NapocaRomania
  3. 3.Department of Infectious Diseases, Faculty of Veterinary MedicineUniversity of Agricultural Sciences and Veterinary MedicineCluj-NapocaRomania
  4. 4.Department of Internal Medicine, Faculty of Veterinary MedicineUniversity of Agricultural Sciences and Veterinary MedicineCluj-NapocaRomania
  5. 5.Instituto de Investigação e Inovação em SaúdeUniversidade do PortoPortoPortugal
  6. 6.Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP)PortoPortugal
  7. 7.Escola Superior Agrária do Instituto Politécnico de Viana do CasteloPonte de LimaPortugal
  8. 8.Department of Veterinary Sciences and Public HealthUniversity of MilanMilanItaly

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