Pathology in Practice

Elvio Lepri Department of Veterinary Medicine, University of Perugia, 06126 Perugia, Italy.

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Francesca Beccati Department of Veterinary Medicine, University of Perugia, 06126 Perugia, Italy.

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Arianna Miglio Department of Veterinary Medicine, University of Perugia, 06126 Perugia, Italy.

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Fabrizio Passamonti Department of Veterinary Medicine, University of Perugia, 06126 Perugia, Italy.

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Fabrizia Veronesi Department of Veterinary Medicine, University of Perugia, 06126 Perugia, Italy.

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Maria Teresa Mandara Department of Veterinary Medicine, University of Perugia, 06126 Perugia, Italy.

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History

A 10-year-old 480-kg (1,056-lb) Italian Warmblood gelding was evaluated because of sudden-onset lateral deviation of the neck. The owners and the referring veterinarian reported that the horse had abnormal rigidity in neck flexion during work on the right rein while being ridden, which started 7 days before admission, and developed progressive right lateral deviation of the neck. The horse was regularly vaccinated against influenza and tetanus and treated alternately with ivermectin and with a combination of ivermectin and praziquantel every 3 months.

Clinical and Gross Findings

On arrival at the hospital, the horse was alert and responsive; heart and respiratory rates were within reference ranges, and rectal temperature was mildly high (38.4°C [101.1°F]). However, the horse leaned its right side against the wall to help itself walk and had a severely ataxic gait. Neurologic examination revealed normal behavior and mental status, with intermittent sensory blunting. The horse had right lateral deviation of the neck, which became more prominent when the horse was blindfolded, and a right head tilt; the left eye had mydriasis, ventral strabismus, absence of pupillary light reflex (direct and consensual), photophobia, and reduction of the menace reflex. Facial sensation was completely absent on the right side, as was the facial and the cervical component of the cervicofacial response. The horse kept its hind limbs placed well forward, with a wide-based stance of the forelimbs.

Standard radiographic views of the head and proximal portion of the neck were unremarkable, as were results of hematologic and serum biochemical analyses and testing for serum antibodies against West Nile virus and equine herpesvirus 1 and 4. The presence of equine herpesvirus 1 in CSF and blood samples was also investigated via PCR assays, and results were negative.

Owing to the horse's poor prognosis and a lack of response to treatment, the owners requested euthanasia. Just prior to euthanasia (by means of IV injection of detomidine, ketamine hydrochloride, and a mixture of embutramide, mebenzonium iodide, and tetracaine hydrochloride), a CSF sample was collected from the C1–2 intervertebral space under ultrasound guidance as described previously,1 and was analyzed within 30 minutes following collection after centrifugation at 600 × g.

At necropsy, several large oval (up to 10 cm in length) white-yellowish fleshy masses were found in the medial portion of the left thigh and lateral to the penis, corresponding to the left superficial inguinal lymph nodes. Similar focal but smaller lesions (1 to 2 cm in diameter) were also observed in both renal cortices. The heart was moderately enlarged with right ventricular distention; in the myocardium of the interventricular septum, just below the aortic ostium, a single large (3 × 4 cm), well-demarcated yellow nodule was present (Figure 1). No gross lesions were observed in the CNS, except for small brownish discolored areas in the cerebellopontine region and in the medulla oblongata.

Figure 1—
Figure 1—

Photograph of the sectioned heart of a horse that was evaluated because of abnormal rigidity in neck flexion of 7 days' duration that progressed to right lateral deviation of the neck. The horse did not respond to treatment and was euthanized. Notice the large well-demarcated granuloma in the upper portion of the interventricular septum, just below the aortic ostium.

Citation: Journal of the American Veterinary Medical Association 251, 10; 10.2460/javma.251.10.1153

Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page→

Histologic Findings

Histologic examination of the renal, nodal, and myocardial masses as well as portions of the CNS and other tissues (ie, liver, lungs, intestines, eyes, and spleen) was performed. Histologically, all lesions were composed of aggregates of multinucleated giant cells and epithelioid macrophages with interspersed lymphocytes and plasma cells, and occasionally associated with sections of adult and larval nematodes. Similar parasitic specimens were also observed in the CNS samples (Figure 2); the nematodes were located adjacent to parenchymal vessels within small necrotic foci and affected the cerebral cortex, basilar nuclei, thalamus, cerebellum, and brainstem. The lesions were characterized by gliosis, gitter cell infiltration, and occasional lymphocytes and plasma cells. In some areas, the inflammatory reaction assumed a more obvious granulomatous or eosinophilic pattern. Scattered foci of eosinophilic and lymphocytic infiltration were also observed in the Virchow-Robin space of arachnoid blood vessels. The lesions were not observed in the spinal cord. In the heart, the lesions included the large, grossly evident, well-demarcated mass with numerous Anitschkow cells; the mass was associated with smaller scattered infiltrative granulomas dispersed between ventricular myocardial cells. The inguinal masses were lymph nodes that had been completely effaced by granulomatous inflammation, with minimal residues of cortical lymphatic tissue.

Figure 2—
Figure 2—

Photomicrograph of a section of cerebral tissue obtained from the horse in Figure 1. There is an area of malacia associated with larvae colonization (arrow). A marked endothelial reaction with early leukocyte and macrophage infiltration is evident. H&E stain; bar = 100 mm.

Citation: Journal of the American Veterinary Medical Association 251, 10; 10.2460/javma.251.10.1153

Findings of CSF Analysis

The CSF sample was clear and slightly xanthochromic. The refractive index (1.005; reference range, 1.004 to 1.006), pH (7.5; reference range, 7 to 9), and protein concentration (52 mg/dL; reference range, 20 to 80 mg/dL) of the CSF sample were considered normal. The RBC and WBC counts were slightly and markedly high, respectively (RBC count, 120 RBCs/μL [reference range, 0 to 100 RBCs/μL]; WBC count, 370 WBCs/μL [reference range, 0 to 8 WBCs/μL]). A differential cell count and cell identification were performed on cytocentrifugated and air-dried CSF smears stained with May-Grünwald-Giemsa stain. Marked cellularity and blood contamination were confirmed. Most of the cells were reactive, occasionally binucleated monocytes and macrophages (87%; reference range, 25% to 35%) with vacuolated cytoplasm containing phagocytosed cellular debris. A small number of lymphocytes (8%; reference range, 70% to 80%), nondegenerated neutrophils (5%; reference range, 0% to 10%), and occasional ependymal cells were also present. Moreover, a few rhabditiform nematodes were observed (Figure 3). Bacteriologic culture of the CSF sample did not yield any bacterial growth, thereby ruling out infection with Listeria spp, which was also considered among the differential diagnoses.

Figure 3—
Figure 3—

Photomicrograph of a cytologic preparation of an antemortem CSF sample collected from the C1–2 intervertebral space of the horse in Figure 1. A few rhabditiform nematodes were detected in the CSF sample, one of which is illustrated here. May-Grünwald-Giemsa stain; bar = 50 μm.

Citation: Journal of the American Veterinary Medical Association 251, 10; 10.2460/javma.251.10.1153

Additional Laboratory Findings

To characterize the morphological features of the recovered nematodes, small pieces of fresh nodal tissue were placed in PBS (0.8% NaCl) solution (pH, 7.4) to allow the migration of the parasites. The larvae collected were examined by light microscopy (400× magnification) after Lugol staining, and identified as Halicephalobus gingivalis on the basis of their morphological and morphometric features2,3 (Figure 4). Furthermore, to confirm the species identification, genomic DNA was extracted both from a pool of 15 specimens isolated from the tissues and from the cytocentrifugated CSF sample resuspended in PBS solution by means of specific protocols for tissue and CSF, respectively, and a commercial kit.a The DNA samples extracted were submitted to a PCR protocol for the amplification of a 670-base pair fragment of the nuclear large subunit ribosomal RNA gene, according to Nadler et al.4 The PCR products obtained were sequenced in both directions, and the DNA sequence was aligned by use of commercial softwareb with sequences of representative free-living and parasitic isolates, according to Nadler et al.4 Both the CSF and tissue-derived aligned sequences, analyzed by the neighbor-joining method with a specific software package,c revealed that the isolates formed a clade with H gingivalis isolates (the highest identity [99.8%] was observed with a Japanese strain [GenBank accession No. AB288935.1]).

Figure 4—
Figure 4—

Photomicrographs of parasites that were present in small pieces of fresh nodal tissue collected from the horse in Figure 1. The larvae were identified as Halicephalobus gingivalis on the basis of their morphological and morphometric features. A—Adult stage of H gingivalis. Lugol stain; bar = 50 μm. B—Dorsal reflection of the ovary is evident. Lugol stain; bar = 20 μm. C—The parasite has a rhabditiform esophagus with isthmus and bulb. Lugol stain; bar = 20 μm.

Citation: Journal of the American Veterinary Medical Association 251, 10; 10.2460/javma.251.10.1153

Morphologic Diagnosis and Case Summary

Morphologic diagnosis and case summary: parasitic meningoencephalitis caused by H gingivalis, associated with systemic spread of the parasite, especially to the kidneys, myocardium, and inguinal lymph nodes in a horse.

Comments

Halicephalobus gingivalis (syn Halicephalobus deletrix) is a small, free-living saprophagous nematode belonging to the order Rhabditada (Family Panagrolaimidae) that is found worldwide. This nematode is sporadically associated with opportunistic infections of horses and humans, frequently with a fatal outcome;5,6 the infective organisms are consistent in their CNS localization. To date, approximately 50 cases of equine infection and 4 cases of human infection have been reported in the medical literature.3 The route of entry of H gingivalis in vertebrate hosts is still unclear. In humans, contamination of skin wounds by manure containing free-living parasites seems the most likely means of infection7; in horses, the most common site of primary infection is probably the oral cavity, wherein the parasite passes through small lacerations of the oral mucosa.6 In some equine cases, the only reported lesion was granulomatous posthitis, suggesting a possible transcutaneous route of entry in the genital region.8,9 In the horse of the present report, neither skin wounds nor cutaneous granulomas were present; however, the severe involvement of inguinal superficial lymph nodes suggested the possibility of a transcutaneous route via the external genitalia or preputial skin. After entry to the host's body, hematogenous spread to highly vascularized tissues occurs, followed by secondary localization. Most of the cases reported in the veterinary medical literature describe lesions in the CNS (both brain and spinal cord) and kidneys and less commonly in the eyes, testes, mammary glands, bones, gingivae, liver, and stomach.10 In the horse of the present report, the lesions were confined to intracranial portions of the CNS, lymph nodes, kidneys, and, interestingly, the heart. Cardiac involvement in cases of halicephalobiasis is considered to be uncommon, and we are aware of only 1 report11 that describes a gray raised lesion (2.5 × 1.5 × 1 cm) beneath the endocardium of the right ventricle in the lateral wall, 5 cm from the atrioventricular valves, in a horse. In a case of human infestation, a single nematode was found in the myocardium with no inflammatory response.5 In the horse of the present report, a large (3 × 4-cm) granulomatous lesion was present in the myocardium together with microscopic scattered multifocal microgranulomas.

Diagnosis of H gingivalis infection is often made after necropsy because of the difficulty in intravitam collection of samples from internal lesions. However, intravitam diagnosis can be achieved by microscopic examination of tissue biopsy specimens obtained from external (mostly gingival) lesions12,13 or, rarely, by percutaneous biopsy of the kidneys.14 To date, H gingivalis has not been observed in samples of body fluids (CSF, blood, saliva, and joint fluid)6 except for urine and semen.14

Verminous encephalomyelitis is a rare cause of neurologic disease in horses. Parasites that can invade the CNS during aberrant migration include strongyloid nematodes (Strongylus equinus, Strongylus vulgaris, and Angiostrongylus cantonensis), Draschia megastoma, Setaria spp, and Hypoderma spp; the most common is H gingivalis.10 In horses with verminous encephalomyelitis, antemortem diagnosis is often impossible, even if suspicion is raised by the sudden onset and rapid progression of clinical signs and the results of CSF analysis.10

In horses with halicephalobiasis, the CSF changes are nonspecific and are represented by high total protein concentration and mild to marked pleocytosis, with high numbers of mononuclear cells and lower numbers of nondegenerate neutrophils and lymphocytes15–20; eosinophils are reported to be rare15 to prevalent, representing up to 42% of total nucleated cells.16 In the horse of the present report, most cells in the CSF sample were mononuclear cells, and eosinophils were rare. The presence and detection of the parasite in a CSF sample are considered exceptional, and can follow massive CNS tissue necrosis in the subependymal areas or CSF contamination by circulating larvae. In this horse, the larvae probably represented extension from cerebral lesions.

In the CSF sample collected from the horse, parasite specimens and parasite DNA were recovered by cytologic and biomolecular investigations, respectively. Recently, a DNA-based approach has been reported to be reliable for parasite identification at the species level and for phylogenetic analysis4; nevertheless, the diagnosis of H gingivalis infection is mostly based on physical characteristics of the parasites, which have specific morphometric and morphological features. The observation of parasite larvae in the CSF sample obtained from the horse of the present report has suggested that intravitam diagnosis is possible, which would then lead to appropriate treatment with specific anthelmintic drugs. However, the susceptibility of H gingivalis to the most common drugs remains to be assessed.

The case described in the present report has highlighted the importance of including verminous meningoencephalitis caused by H gingivalis among the differential diagnoses for neurologic diseases in horses, and the potential value of CSF analysis for determination of a definitive diagnosis. A feature of this case that is worthy of note was the massive myocardial lesion, which has not been previously reported in cases described in the literature, to our knowledge.

Footnotes

a.

QIAmp DNA Mini Kit, QIAGEN Spa, Milan, Italy.

b.

ClustalW, EMBL-EBI, Hinxton, Cambridgeshire, England. Available at: www.ebi.ac.uk/Tools/msa/clustalw2/. Accessed Sep 10, 2013.

c.

MEGA 5. Available at: www.megasoftware.net/. Accessed Sep 12, 2013.

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