Signalment:  

14-year-old, thoroughbred, gelding, horse, (Equus caballus).The patient was admitted for evaluation of chronic colic, and had a distended abdomen on arrival. The horse was diagnosed with uroperitoneum and was euthanized after a tear in the ventral portion of the urinary bladder was confirmed on abdominal ultrasound and cystoscopy. Clinical signs of disease involving other organ systems (e.g. respiratory tract) were not reported.

Gross Description:  

The patient was in satisfactory nutritional and good postmortem condition. The peritoneum contained abundant turbid, light-yellow fluid with a distinct ammonia odor (i.e. urine), and was hyperemic with adherent plaques of fibrin. The urinary bladder was distended and a 7 x 5 cm thin, gray-green and friable (necrotic) focus within the ventral wall was confirmed. However, examination of the lungs revealed multifocal to coalescing 0.5- 5 cm diameter firm tan nodules that extended into all fields, replacing the pulmonary parenchyma and elevating the visceral pleura (figure 1). Intervening pulmonary parenchyma was pale pink to dark red and mildly firm cranioventrally. The brain, spinal cord, and remainder of the thoracic and abdominal viscera were grossly unremarkable.

Histopathologic Description:

Approximately 80% of the pulmonary parenchyma was effaced by multifocal to coalescing nodules composed of proliferating spindle cells (fibroblasts) embedded within a fibrillar eosinophilic (collagenous) stroma, and infiltrated by large numbers of lymphocytes and histiocytes, with fewer neutrophils. Occasionally, alveolar macrophages contained 3-4μm diameter eosinophilic intranuclear inclusion bodies that peripheralized the chromatin (Cowdry Type A inclusions; figure 2 (arrow)). Within affected regions, the majority of alveoli were lined by plump cuboidal epithelial cells (bronchiolization). Affected alveoli, and occasional bronchi and bronchioles, were filled with numerous foamy macrophages, neutrophils, and cell debris. Within unaffected regions of lung, alveoli were often ectatic (hyperinflated), and there was mild multifocal alveolar hemorrhage.

Morphologic Diagnosis:  

Lung: severe chronic multifocal–coalescing sclerosing lymphohistiocytic and neutro-philic bronchointerstitial pneumonia with intrahistiocytic intranuclear inclusion bodies (consistent with equine multinodular pulmonary fibrosis).

Lab Results:  

None.

Condition:  

Equine multinodular pulmonary fibrosis, EHV-5

Contributor Comment:  

Equine multinodular pulmonary fibrosis (EMPF), one of the relatively few differentials for interstitial lung disease in the horse, results in a unique gross and histologic appearance dominated by a nodular pattern of marked interstitial fibrosis.¹⁰ Clinical signs of affected horses may include tachypnea, dyspnea, nasal discharge, coughing, lethargy, inappetence, and poor body condition. Affected horses may have increased bronchovesicular sounds, fever, and/or hypoxemia on clinical exam.¹⁴ Hematology typically reveals a neutrophilia (in some cases with increased bands), lymphopenia or lymphocytosis, and monocytosis.^7,14 A nodular interstitial pattern is often identified on thoracic radiographs and ultrasound, resulting in differential diagnoses of EMPF, fungal pneumonia and pulmonary neoplasia. Concurrent with the interstitial pulmonary disease, thoracic ultrasonography may also reveal pulmonary artery dilation, consistent with pulmonary hypertension. Transtracheal wash fluid is predominantly neutrophilic (degenerate or non-degenerate), with fewer macrophages.¹⁴ No breed or sex predilection has been established, and although affected horses are typically middle-aged or geriatric, cases have been reported in horses as young as four years-old.¹²

Two gross patterns of EMPF have been described, both of which are characterized by multifocal moderately firm, tan, bulging nodules throughout the pulmonary parenchyma. In the more common of the two gross forms (the form exhibited by this horse), these nodules are numerous but small (up to 5 cm in diameter), and coalesce, often resulting in scant intervening parenchyma. In the second gross pattern, the nodules are less frequent, larger (up to 10 cm in diameter), and discrete, with the intervening pulmonary parenchyma largely unaffected.¹² Histologically, both gross forms are characterized by extensive interstitial deposition of mature collagen, accompanied by moderate mixed inflammatory infiltrates, consisting pre-dominantly of lymphocytes, macrophages and neutrophils. Alveoli in affected regions are typically lined by plump, cuboidal epithelium, and airways contain abundant neutrophils and macrophages. Alveolar macrophages occasionally contain ampho-philic to eosinophilic intranuclear inclusion bodies. Less frequently, the nodules consist of dense sheets of disorganized collagen that completely efface the normal alveolar pattern.¹²

Equine herpesvirus-5 (EHV-5) is a gamma herpesvirus strongly associated with EMPF, and can be identified by polymerase chain reaction (PCR), immunohistochemistry (IHC), in situ hybridization, virus isolation, and transmission electron microscopy (TEM).^2,7,12-14 Although causality has not been proven by meeting all of Koch’s postulates, the main histologic and immunohistochemical features have been recapitulated in horses inoculated with EHV-5 isolated from EMPF-affected horses.¹³ However, because inoculated horses lacked clinical signs associated with EMPF, were PCR-negative for the inoculated EHV-5 viruses (within the lungs), and the virus was unable to be re-isolated, the authors suggest that the immuno-histochemical detection of virus in the inoculated horses having deposition of fibrous connective tissue likely represented a pre-clinical, yet latent, phase of viral infection.¹³ Further supporting EHV-5 as an important etiologic factor in the development of EMPF, quantitative real-time PCR performed in an affected horse revealed higher viral load within the lungs than in other tissues, with the highest load within the most severely affected/fibrotic regions of lung.⁴ In addition, gamma herpesviruses have been associated with fibrotic lung diseases in other species, including a variably fibrotic broncho-interstitial pneumonia with syncytial cell formation in donkeys associated with asinine herpesvirus-4 and 5 (AHV-4 and 5),³  and human idiopathic pulmonary fibrosis associated with human herpesvirus-8 (HHV-8) and Epstein-Barr virus.^10,11 Moreover, mice that are knockouts for the IFNγ cytokine receptor (an important mediator of the Th1 antiviral response), develop progressive pulmonary interstitial fibrosis after chronic infection with murine gamma herpesvirus-68.^5,11 Although the precise mechanisms of gamma herpesvirus-induced fibrosis and immune system evasion in most species are unknown, they are likely related to the specific viral-induced cytokine, chemokine, and/or receptor profile within the host, which either directs the immune system toward a Th2 response (thereby inhibiting a Th1 response and facilitating fibrosis), or at least prevents an effective Th1 antiviral response.^6,11 Such mechanisms have already been established in the study of human gamma herpesviruses, including viral CCL-1 (vCCL-1), vCCL-2, and vCCL-3 encoded by HHV-8 that activate Th2 cell chemokine receptors (CCR8, CCR3 and CCR8, and CCR4, respectively), and a viral IL-10 homologue produced by human Epstein-Barr virus that results in inhibition of the Th1 antiviral response.^6,11 In spite of supporting evidence of EHV-5 infection inciting EMPF, EHV-5 may not be the sole cause. As with many infectious diseases, concurrent viral infections (such as EHV-2 and AHV-4 or 5), and the host’s immune status, may also be important contributors to the pathogenesis of EMPF.^1,5,11,12

Although successful treatment with corticosteroid and antiviral (acyclovir) therapy is reported, overall, response to treatment is variable and the prognosis of this disease is generally considered poor.¹⁴ This case was unusual in that there were no reported clinical signs that were clearly attributable to respiratory disease. However, a preclinical, latent phase of infection is considered unlikely given the presence of numerous inclusion bodies. Although the uroperitoneum accounted for the most recent episode of colic, it is possible that the pneumonia caused chronic lethargy and inappetence, vague signs that were interpreted as chronic colic. Regional trans-mural necrosis of the urinary bladder was confirmed histologically, and was attributed to pressure-induced ischemia secondary to distension. As there was no evidence of urethral obstruction, a neurogenic cause was suspected. However, no histologic lesions were identified within the spinal cord.

JPC Diagnosis:  

Lung: Fibrosis, interstitial, nodular, multifocal to coalescing, severe with lymphohistiocytic interstitial inflammation, alveolar neutrophilic and histiocytic exudate, type II pneumocyte hyperplasia and histiocytic intranuclear viral inclusion bodies, thoroughbred, Equus caballus.

Conference Comment:  

We thank the contributor for both an excellent example and thorough summary of equine multinodular pulmonary fibrosis (EMPF) in horses. This entity has also been extensively reviewed in previous Wednesday Slide Conferences (2011 Conference 1 Case 2 2012 Conference  24 Case 3 2013 Conference 18 Case 1 , but it was chosen again because of its highly distinctive and unique histomorphology.  Participants identified multifocal discrete lung nodules with abundant interstitial fibrosis, marked type II pneumocyte hyperplasia, and irregular alveolus-like spaces filled with an inflammatory exudate composed of neutro-phils, fibrin, and alveolar macro-phages which occasionally contain a 2-4 um magenta intranuclear inclusion body. Conference participants also noted especially prominent pleural arteries in areas adjacent to the nodules of fibrosis with hypertrophic smooth muscle in the tunica media indicative of pulmonary hypertension associated with pulmonary fibrosis.

As mentioned by the contributor, gamma herpesviruses have been associated with progressive pulmonary fibrotic disorders in humans, donkeys, horses, and rodents. In dogs, canine idiopathic pulmonary fibrosis is a progressive pulmonary fibrotic disorder predominantly in aged West Highland white terriers (WHWT).⁹ Recently, investigators have tried to elucidate a relation between this disorder in WHWTs and gamma-herpesvirus infection; however, no evidence a connection was found. Given this condition’s predilection for WHWT, it is thought that there is a genetic component to this disease in this breed of dog rather than an infectious etiology.⁹

In addition to pulmonary fibrosis, conference participants discussed the association of a gamma herpesvirus with retroperitoneal fibromatosis (RF), an aggressive proliferation of highly vascular fibrous tissue subjacent to the peritoneum involving the ileocecal junction and mesenteric lymph nodes in rhesus macaques.¹⁰ RF is associated with co-infection of simian retrovirus 2 (SIV) causing simian acquired immunodeficiency syndrome (SAIDS) and RF-associated herpesvirus (RFHV). This condition is closely related to Kaposi’s sarcoma in humans, caused by co-infection of human herpesvirus 8 (HHV8) and human immuno-deficiency virus (HIV), and is one of the first illnesses associated with the development of AIDS.¹⁰ Additionally, rhesus macaque rhadinovirus, another gammaherpesvirus, is also closely related to HHV8 and both have been associated with the development of B-cell lymphoma.⁸

References:

1. Back H, Kendall A, Grandón R, et al. Equine multinodular pulmonary fibrosis in association with asinine herpesvirus type 5 and equine herpesvirus type 5: a case report. Acta Vet Scand. 2012;54(57):1-5.
2. Caswell JL, Williams KJ: Equine multinodular pulmonary fibrosis. In: Maxie MG ed. Jubb, Kennedy, and Palmer’s pathology of domestic animals. Vol 2. 6^th ed. St. Louis, Missouri: Elsevier; 2016:568-569.
3. Kleiboeker SB, Schommer SK, Johnson PJ, et al. Association of two newly recognized herpesviruses with interstitial pneumonia in donkeys (Equus asinus). J Vet Diagn Invest. 2002;14:273-280.
4. Marenzoni ML, Passamonti F, Lepri E, et al. Quantification of Equid herpesvirus 5 DNA in clinical and necropsy specimens collected from a horse with equine multinodular pulmonary fibrosis. J Vet Diagn Invest. 2011;23(4):802-806.
5. Mora AL, Woods CR, Garcia A, et al. Lung infection with γ-herpesvirus induces progressive pulmonary fibrosis in Th2-biased mice. Am J Physiol Lung Cell Mol Physiol. 2005;289:L711-L721.
6. Nicholas J. Human gamma-herpesvirus cytokines and chemokine receptors. J Interf Cytok Res. 2005;25:373-383.
7. Niedermaier G, Poth T, Gehlen H. Clinical aspects of multinodular pulmonary fibrosis in two warmblood horses. Vet Rec. 2010;166:426-430.
8. Orzechowska BU, Powers MF, et al. Rhesus macaque rhadinovirus-associated non-Hodgkin lymphoma: Animal model for KSHV associated malignancies. Blood. 2008; 112:4227-4234.
9. Roels E, Dourcy M, et al. No evidence of herpesvirus infection in West Highland white terriers with canine idiopathic pulmonary fibrosis. Vet Pathol. 2016; 53(6):1210-1212.
10. Rose TM, Strand KB, et al. Identification of two homologs of the Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) in retroperitoneal fibromatosis in different macaque species. J Virol. 1997; 71:4138-4144.
11. Tang Y, Johnson JE, Browning PJ, et al. Herpesvirus DNA is consistently detected in lungs of patients with idiopathic pulmonary fibrosis. J Clin Microbiol. 2003;41(6):2633-2640.
12. Williams KJ. Gammaherpesviruses and pulmonary fibrosis: evidence from humans, horses and rodents. Vet Pathol. 2014;51(2):372-384.
13. Williams KJ, Maes R, Del Piero F, et al. Equine multinodular pulmonary fibrosis: a newly recognized herpesvirus-associated fibrotic lung disease. Vet Pathol. 2007;44:849-862.
14. Williams KJ, Robinson NE, Lim A, et al. Experimental induction of pulmonary fibrosis in horses with gammaherpesvirus Equine Herpesvirus 5. PLoS ONE. 2013;8(10):e77754, doi. 10.1371/journal.pone.0077754.
15. Wong DM, Belgrave RL, Williams KJ, et al. Multinodular pulmonary fibrosis in five horses. J Am Vet Med Assoc. 2008;232(6):898-905.

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2-1. Lung, horse.

2-2. Lung, horse.

2-3. Lung, horse.

2-4. Lung, horse.

2-5. Lung, horse.

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