Pathology in Practice

Marta Polinas 1Dipartimento di Medicina Veterinaria, Università degli Studi di Sassar, 07100 Sassari, Italy.

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Elisabetta Antuofermo 1Dipartimento di Medicina Veterinaria, Università degli Studi di Sassar, 07100 Sassari, Italy.

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Sara Boccuni 1Dipartimento di Medicina Veterinaria, Università degli Studi di Sassar, 07100 Sassari, Italy.

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Silvia Dei Giudici 2Istituto Zooprofilattico Sperimentale della Sardegna G Pegreffi, Via Vienna 2, 07100 Sassari, Italy.

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Tiziana Cubeddu 1Dipartimento di Medicina Veterinaria, Università degli Studi di Sassar, 07100 Sassari, Italy.

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Giovanni P. Burrai 1Dipartimento di Medicina Veterinaria, Università degli Studi di Sassar, 07100 Sassari, Italy.
1Dipartimento di Medicina Veterinaria, Università degli Studi di Sassar, 07100 Sassari, Italy.
2Istituto Zooprofilattico Sperimentale della Sardegna G Pegreffi, Via Vienna 2, 07100 Sassari, Italy.

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History

A 4-year-old 15.3-kg (33.7-lb) sexually intact male mixed-breed dog, which was vaccinated against canine distemper virus, canine adenovirus type 2, canine parvovirus, canine parainfluenza virus-2, and Leptospira spp, was lost by the owner after a boar-hunting trip in the countryside of Sardinia, Italy. After 4 days, the dog came back home but in the interim had developed intense facial pruritus and episodes of emesis; the vomited material contained hair, which was presumed to be of porcine origin.

Clinical and Gross Findings

At a clinical evaluation, the dog had signs of depression and high heart and respiratory rates. The dog's rectal temperature was high (39.8°C [103.6°F]). Treatment with an antihistamine and a corticosteroid was administered to control the intense pruritus associated with the dog's right cheek area and lips. After a few hours, copious drooling, seizures, and generalized fasciculation were observed. Control of the neurologic signs (administration of anticonvulsants and muscle relaxants) was attempted with no clinical benefit. After 24 hours, the dog died, and a necropsy was performed at the University of Sassari's veterinary teaching hospital.

On macroscopic examination, severe hyperemia and abrasions of the skin in the temporal region associated with edematous swelling of the right cheek (Figure 1) were evident. The left side of the head was unaffected. Hemorrhagic areas and petechiae were detected in the right gingival mucosa, peritoneum, and pleura. The endocardium contained locally extensive areas of hemorrhage, which were particularly severe at the level of the atrioventricular valves. The meninges appeared diffusely congested and slightly edematous. Severe congestion was particularly evident on the right occipital lobe of the cerebrum.

Figure 1—
Figure 1—

Photographs obtained during necropsy of a 4-year-old mixed-breed dog that became lost after being used for boar hunting in the countryside of Sardinia, Italy. After 4 days, the dog returned home but had developed intense facial pruritus and episodes of emesis; the vomited material contained hair, which was presumed to be of porcine origin. A—Severe and locally extensive skin hemorrhagic abrasions (apparently caused by self-trauma) are present around the right eye and muzzle. Inset—Multifocal petechiae are visible in the gingival mucosa. B—Locally extensive areas of moderate hemorrhage are evident in the left atrioventricular valve. C—The brain has severe and extensive congestion of the meninges and right occipital lobe. D—In the gastrointestinal tract, there is evidence of multifocal to locally extensive, severe, acute hemorrhagic gastroenteritis associated with infestations of tapeworms and nematodes.

Citation: Journal of the American Veterinary Medical Association 254, 12; 10.2460/javma.254.12.1411

The stomach contained a moderate amount of partially digested blood admixed with filamentous material of vegetal origin, bristles of wild boar, and numerous adult ascarids. The gastric mucosa had multifocal, round, red to blackish craterlike areas measuring 1.2 cm in diameter, consistent with gastric ulcers. The small intestine had moderate to severe segmental hemorrhagic enteritis with numerous tapeworms, hookworms, and roundworms. The liver appeared enlarged, and the gallbladder was moderately distended. The lungs were severely and diffusely edematous and congested.

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

Histopathologic Findings

Samples of tissues of interest were fixed in neutral-buffered 10% formalin, embedded in paraffin, and stained with H&E stain. Microscopic examination of sections of the brain revealed diffuse congestion and shrunken, hypereosinophilic neurons often with pyknotic nuclei (necrotic neurons) in the pons (brainstem [Figure 2]); the neurons had oval, 4- to 6-μm-long, homogeneous eosinophilic intranuclear inclusion bodies that marginated the chromatin. In the same anatomic location, focal microaggregates of monocytes and lymphocytes were arranged around vascular walls and expanded the Virchow-Robin spaces. Nodular gliosis and scattered neutrophils with fragmented nuclei (karyorrhexis) were present. Multifocal, subendocardial (associated with the atrioventricular valves) and ventricular myocardial hemorrhages, interstitial edema, and slightly dilated lymphatic vessels were found in the heart. Some tissue samples (brain, cerebellum, lungs, heart, kidneys, tonsils, stomach, and spleen) were submitted for molecular evaluation to identify the presence of pseudorabies virus (PRV). A nested PCR assay that targeted a fragment of the PRV genome encoding the glycoprotein B was performed, as described by Balasch et al.1 The DNA extraction from formalin-fixed, paraffin-embedded tissue samples was performed with a kita in accordance with the manufacturer's instructions.

Figure 2—
Figure 2—

Photomicrographs of sections of brain tissue from the dog in Figure 1. A—Notice that neurons in the brain are shrunken and hypereosinophilic (arrows) and surrounded by few glial cells (satellitosis). H&E stain; bar = 10 μm. Inset—The neurons in the brain contain 4- to 6-μm-long eosinophilic nuclear inclusion bodies. H&E stain; bar = 10 μm. B—The Virchow-Robin space is expanded by several layers of lymphocytes. H&E stain; bar = 10 μm.

Citation: Journal of the American Veterinary Medical Association 254, 12; 10.2460/javma.254.12.1411

In the PCR assay reactions, a negative control (purified PCR-grade water) and a positive control (wild strain PRV that had been previously isolated and characterized by the Istituto Zooprofilattico Sperimentale della Sardegna) were included. The PCR assay products were visualized with UV light in a 2% agarose gel stained with a fluorescent nucleic acid dyeb (Figure 3). Pseudorabies virus was detected only in the brainstem samples that had histologically detectable lesions. Results of a reverse transcription PCR assay for canine distemper virus detection performed on brain and lung samples were negative.

Figure 3—
Figure 3—

Results of agarose gel electrophesis of PRV-specific PCR assay products obtained from the dog's brainstem tissue samples that had undergone PRV-specific PCR assay. Notice the expected product size of 195 bp, consistent with a diagnosis of pseudorabies (Aujesky disease). MW = Molecular-weight marker lane. Lane 1 = negative control sample (purified PCR-grade water). Lane 2 = sample from the pons (brainstem). Lane 3 = positive control sample (previously isolated and characterized as wild strain PRV).

Citation: Journal of the American Veterinary Medical Association 254, 12; 10.2460/javma.254.12.1411

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: moderate to severe, acute, multifocal lymphohistiocytic meningoencephalitis with neuronal necrosis and eosinophilic intranuclear inclusion bodies.

Case summary: pseudorabies in a hunting dog.

Comments

Pseudorabies, also known as Aujeszky disease, is a contagious and worldwide economically important disease caused by Suid herpesvirus 1 (SuHV-1), a member of the Alphaherpesvirinae subfamily.2 Pseudorabies is a reportable infectious disease in the United States. Pigs and wild boars represent the natural hosts for the virus, but other species, such as cattle, sheep, goats, horses, dogs, and cats can occasionally be infected.3 Pseudorabies has spread almost globally since the early 1980s, probably because of the emergence of more virulent strains and the industrialization of pig farming.4 Crowding conditions in confined spaces, which are characteristic of intensive pig farming, favor the diffusion of the infection in swine populations.4 Although pseudorabies has been eradicated from domestic pig populations in many countries, PRV is still detected in wild boar populations and in hunting dogs, which allows virus permanence in certain areas.5,6 In Italy, despite eradication efforts since 1997, PRV is still present in pig farms, and wild suids represent a reservoir of disease.3

In domestic pigs and wild boars, PRV may be transmitted by nose-to-nose contact, transplacentally, or during coitus.7 Viral replication takes place in cells of the respiratory tract from which the virus reaches the neurons of the facial and pharyngeal area (olfactory, trigeminal, and glossopharyngeal nerves). Centripetal spread of the virus from the peripheral nerves progresses to the spinal cord or brain.8,9 Among affected suids, various clinical and pathological features are observed depending on the viral strain virulence and an animal's age and immune status. Furthermore, nonspecific clinical signs, such as signs of depression, anorexia, fever, and respiratory distress are frequently detected in pigs with pseudorabies; abortion in infected sows may occur. In young piglets, the nervous system is severely affected following infection with development of ataxia, tremors, opisthotonos, and epileptic seizures.10 Swine and wild boars represent a reservoir for the virus and can develop a latent phase of the infection, during which the virus localizes at the level of the trigeminal ganglia or sacral ganglia, olfactory bulb, and tonsils.9,10

In pigs with pseudorabies, endothelial cells, lymphocytes, macrophages, and epithelia are viral replication sites.10 Other animals, such as dogs, usually become infected through contact with infected pig carcasses and by ingestion of raw meat or offal from pigs.9 Following oronasal transmission of infection in accidental hosts other than swine, PRV mostly localizes in the nervous system as a result of retrograde axonal transport. Infected dogs develop frantic behavior, mandibular paralysis, and copious drooling and usually die within 7 days after the onset of clinical signs.9 The clinical course is usually hyperacute to acute and characterized by hyperthermia and intense, usually unilateral, pruritus attributable to irritation of nerve endings by the virus. The intense pruritus can affect any part of the body, although the head (trigeminal nerve) and the hind limbs are the most commonly affected anatomic regions. Affected animals generally self-traumatize, and continued scraping and scratching can lead to further aggravation of injuries.11

The diagnosis of pseudorabies or Aujeszky disease is based on anamnestic data, clinical signs, and pathological findings. Moreover, clinical and macroscopic changes are nonspecific and sometimes remain unnoticed, especially by the animals’ owners.1

Commonly, the histopathologic changes of pseudorabies are localized to the nervous system. Nonsuppurative meningoencephalitis and ganglioneuritis are usually evident in both gray and white matter with a variable location depending on the virus penetration site.2 Affected areas often reveal perivascular cuffing characterized by monocytes, scattered granulocytes, and sometimes glial nodules. Neuronal necrosis may be focal or disseminated. Trigeminal ganglia are commonly affected by neutrophilic infiltration with scarce lymphocytes and macrophages.7 Subendocardial hemorrhages, skin abrasions, bloody intestinal content, and mesenteric lymphadenopathy are the most frequent necropsy findings for dogs with pseudorabies.12 On the basis of the neurologic signs, differential diagnoses for pseudorabies include other causes of encephalitis, such as infection with other viruses (eg, rabies virus or distemper virus), bacterial agents (eg, Listeria sp or Escherichia coli), fungi (Cryptococcus sp), or protozoa (Toxoplasma gondii or Neospora sp) and exposure to various toxic substances.13

As for the case described in the present report, the diagnosis of pseudorabies is confirmed by virus identification in affected tissue samples, which is accomplished by the use of virus detection techniques, such as immunofluorescence, in situ hybridization, and immunohistochemical analyses. Molecular techniques are widely used and a PRV-specific PCR assay is considered the method of choice for virus identification in tissues.13,14 Although pseudorabies has been eliminated in domestic pigs in many European countries and in the United States, this disease is detected in wild boar populations and dogs used for boar hunting in certain geographic locations.15,16 There is no established treatment for pseudorabies in dogs and, as for the dog of the present report, the disease is fatal.17 The onset of neurologic signs in hunting dogs could be an early indication of the spread of pseudorabies in swine populations on small and backyard farms that have low biosecurity as well as among animals in free-range herds.18,19

Footnotes

a.

QIAamp DNA formalin-fixed paraffin-embedded tissue kit, Qiagen, Milan, Italy.

b.

GelRed, Biotium, Milan, Italy.

References

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