Conference 16 - 2016 Case: 4 20170125

Signalment:

Seven-year-old quarter horse mare (Equus ferus caballus).In June 2015, the horse presented to the referring veterinarian with bilateral conjunctivitis that progressed to severe anterior uveitis in the left eye. Foot abscesses, distal limb cellulitis, mandibular lymphadenopathy, nasal discharge, and hives developed subsequently. Treatments included ceftiofur, oxytetracycline, dexa-methasone, nonsteroidal anti-inflammatory drugs, and a two-week course of doxycycline. Despite treatment, the horse remained hyperfibrinogenemic at 800-1300 mg/dL and developed narcolepsy a few months later. Due to health concerns and the poor prognosis, the horse was euthanized in January 2016 and submitted to Cornell Animal Health Diagnostic Center for necropsy and tissue collection.

Gross Description:

There was approximately 200 mL of yellow tinged transparent fluid (serous effusion) within the peritoneal cavity. The capsular surface of the liver was diffusely thickened, mottled white to tan to purple to black. There were thousands of multifocal to coalescing, generalized, white, 1-3 mm, hard white nodules along the capsular surface with a few dozen similar nodules within the parenchyma. Similar nodules were present in the thymus and surrounding the mediastinal fat and in all lung lobes. These nodules were presumed to be parasitic granulomas, which were confirmed histologically. Evidence of chronic laminitis was present in both forelimbs. The brain was grossly normal.

Histopathologic Description:

Brain, cerebrum, and cerebellum: Diffusely, meninges of the cerebellum and cerebrum are markedly expanded by a dense infiltrate of lymphocytes, macrophages, fewer neutrophils, and rare plasma cells interspersed with rare wispy spirochetal bacteria. Within the neuroparenchyma, the Virchow-Robin spaces of blood vessels are surrounded by a similar inflammatory infiltrate. Blood vessels are often prominent, characterized by endothelial hypertrophy and have variable branching. In the white and grey matters are increased numbers of enlarged glial cells with increased eosinophilic cytoplasm (astrocytes, presumptive). Multifocally within the choroid plexus are clusters of lymphocytes and histiocytes, along with few eosinophils.

Morphologic Diagnosis:

1. Brain, cerebrum, and cerebellum: Severe, multifocal to coalescing, chronic lymphohistiocytic neutrophilic meningo-encephalitis with lymphohistiocytic eosinophilic choroid plexitis, branching blood vessels, astrocytosis, and rare intralesional spirochetal organisms. Other final morphologic diagnoses (slides not included):
2. Lymphohistoicytic meningo-myeloencephalitis and radiculoneuritis of the spinal cord and ganglia, respectively
3. Moderate, multifocal, chronic lymphocytic hypophysitis
4. Multifocal, chronic parasitic granulomas in the liver, lung, and thymus
5. Chronic lymphoplasmacytic portal hepatitis with capsular and bridging fibrosis
6. Multifocal laminitis and chronic foot abscess
7. Lymphoid depletion in spleen and thymus

Lab Results:

Bloodwork in early December 2015 revealed elevated gamma glutamyl transferase (GGT) at 61 U/L (normal range 9-24 U/L), rising globulins at 2.3 g/dL from a previous value of 1.6 g/dL (normal range 2.8-4.7 g/dL), and a lymphocyte count of 1,940 cell/uL, up from a previous count of 1,380 cells/uL (normal range 1,000-4,900 cells/uL; lymphopenia is <1,500 cells/uL).

B cell concentration was markedly decreased at 19 cells/uL of 1,940 total lymphocytes/uL (0.98% B cells), with 1.0% CD19 B cells (median, CI = 9.0%, 2.0%), 0.2% CD21 B cells (median, CI = 10.2%, 4.2%), and 0.9% IgM B cells (median, CI = 10.2%, 2.1%).

The CD4+ and CD8+ T-cell distributions were slightly increased, and the CD4/CD8 ratio was within the normal reference interval.

Serum IgG concentration was markedly decreased at 423 mg/dL (median, CI = 1,760 mg/dL, 603 mg/dL) and serum IgM concentration was within the normal reference interval at 63 mg/dL (median, CI = 100 mg/dL, 50 mg/dL; deficiency is < 25 mg/dL).

Bacterial cultures and virus isolation of brain tissue were both negative. A quantitative PCR for Borrelia burgdorferi yielded a CT value of 32 (positive result).

Condition:

Meningoencephalitis/Mutated feline enteric coronavirus (FIP)

Contributor Comment:

The histochemical stain of a section of brain with Modified Steiner silver stain and immunohistochemical (IHC) stain for Borrelia burgdorferi confirmed spiral organisms within areas of inflammation in the meninges of the cerebellum and cerebrum. IHC stains of section of cerebrum for eastern equine encephalitis virus, equine herpes virus-1, West Nile virus, and rabies virus yielded no immunoreactivity. Inflammatory cells were strongly immuno-reactive to CD3 and IBA1; however, rare CD20 and no Pax5 immunoreactivity were detected, confirming lack of plasma cells in areas of inflammation and consistent with common variable immunodeficiency (CVID).

CVID is a primary immunodeficiency disease of humans and horses that encompass a group of heterogenous disorders characterized by hypo-gammaglobulinemia. Generally, at least two isotypes of antibodies are affected, although IgG deficiency alone is recognized. Human CVID patients often present with recurrent respiratory infections and have a high frequency of autoimmune and lympho-proliferative disease.^1,8,9 It is one of the most common primary immuno-deficiencies reported in humans, with an incidence rate of 1 in 25,000 humans. In horses, CVID is a rare condition with relatively few cases reported,^1,2,3,10,14 though the Equine Immunology Laboratory at Cornell College of Veterinary Medicine has diagnosed this condition in over 50 horses since 2002 and has been actively investigating potential genetic and epigenetic mechanisms of disease.¹² Current research on equine CVID focuses on the disruption of B cell development in the bone marrow, and has identified decreased mRNA expression and incomplete demethylation of the PAX5 gene, required for commitment and differentiation of B cells.^12,13

Like human patients, horses clinically manifest with recurrent infections of the respiratory tract. In addition, persistent bacterial meningitis has been associated with infection by common skin contaminants such as Staphylococcus spp.,^3,10 while Borrelia burgdorferi has been highly suspected in other cases of meningitis. One case report of CNS and PNS inflammation in a CVID horse documented a positive Western blot analysis result with low to moderate Borrelia burgdorferi antibody response in serum and a positive PCR assay result from CSF using primers for the outer surface protein A (ospA) gene.⁵ These tests confirm exposure to the bacterium; however, neither test demonstrates active Borrelia burgdorferi infection within areas of CNS inflammation. Lyme neuroborreliosis in horses, as with most species, is characterized by suppurative or non-suppurative, lymphoplasmacytic, histiocytic perivascular to diffuse inflammation most severely affecting the CNS, including the meninges, ganglia, and cranial and spinal nerve roots, with varying degrees of necrosis, fibrosis, and neuro-parenchymal invasion.⁴

In the present case, the inflammation is predominately lymphocytic and histiocytic and the distribution includes the spinal cord and ganglia, meninges, choroid plexus, pituitary gland, and neuroparenchyma. By histochemistry and immunohistochemistry, rare spirochetal organisms were present within areas of perivascular inflammation, while a quantitative PCR confirmed the presence of Borrelia burgdorferi nucleic acid in the affected cerebrum . The history of uveitis and narcolepsy, the clinical data, histologic findings of severe meningo-myeloencephalitis, choroid plexitis, and hypophysitis, and ancillary testing are consistent with Lyme neuroborreliosis.^4,11 The severe inflammation, fibrosis and parasitic granulomas in the liver, lung, and thymus are attributed to massive parasitic migration; a finding consistent with CVID and a lack of antibody response to parasitic antigens.^2,14 The lack of humoral immunity, the primary host defense mechanism against Borrelia burgdorferi, likely contributed to chronic Lyme disease in this horse with CVID.

JPC Diagnosis:

Cerebrum: Chorio-meningoencephalitis, lymphohistiocytic, multifocal to coalescing, marked, quarter horse, Equus ferus caballus.

Conference Comment:

Lyme neuro-borreliosis is an uncommon manifestation of Lyme disease caused by Borrelia burgdorferi sensu lato infection in the nervous system, and is typically associated with immunosuppression in horses, humans, and experimental laboratory animal models.^4-6 The contributor provides an outstanding demonstration of that patho-genesis in this case of natural infection in a horse with common variable immuno-deficiency (CVID). As mentioned above, CVID is associated with a late-onset B cell lymphopenia and hypo-gammaglobulinemia with marked decrease in serum IgG. CVID typically manifests as opportunistic recurrent pneumonia, septicemia, and meningitis.¹⁴

a White-footed mice are the principal reservoir host for B. burgdorferi in the endemic Northeastern United States, and the bacteria are transferred to susceptible host species by the Ixodes sp. tick vector. B. burgdorferi localizes in the digestive tract of ixodid ticks via its outer surface protein A (OspA) after feeding on an infected reservoir host.⁷ When the vector attaches to a susceptible mammalian host and takes a blood meal, there is a subsequent increase in temperature within the tick digestive tract. This change in temperature represses OspA expression and induces OspC synthesis.

This new conformation allows the spirochete to localize to the salivary glands of the tick. Interestingly, this change in conformation can take as long as 48 hours to complete, necessitating the prolonged attachment of the tick to the host. The spirochete then enters the host via the ticks salivary secretions during feeding.⁷

Previous reports of borreliosis in horses have documented arthritis, uveitis, encephalitis, and ataxia.⁵ Uveitis, present in this case, is the most common reported extra-neural manifestation of B. burgdorferi infection in horses, but is rarely reported in other species.⁶ The most common mani-festation of disease in dogs is polyarthritis, with fewer cases of membranoproliferative glomerulonephritis.^4-6 Equine neuro-borreliosis is challenging to diagnose clinically due to the wide variability in clinical presentation and current lack of reliable antemortem diagnostic tests; however, the conference moderator instructed that the index of suspicion for Lyme disease should be high in horses that present with neurologic deficits and concurrent uveitis.^4-6

Few conference participants included Lyme disease as a differential diagnosis in this case. Most favored a viral encephalitis caused by an alphavirus (EEE, WEE, VEE), rabies, or West Nile virus due to the relatively non-specific lymphohistiocytic inflammation in this case. Others included equine protozoal myelitis caused by Sarcocystis neurona; however, one would expect to see necrotizing granulomatous and eosinophilic lesions, which are not a feature of this case.⁶ In conjunction with the excellent images provided by the contributor, the Joint Pathology Center ran a Warthin-Starry silver stain, which highlights numerous argyrophilic spirochetes consistent with B. burgdorferi within the inflamed neuroparenchyma. This case demonstrates the importance of including Lyme disease as a differential diagnosis in horses with neurologic disease.

References:

1. Ardeniz O, Cunningham-Rundles C. Granulomatous disease in common variable immunodeficiency. Clin Immunol. 2009; 133:198-201.
2. Flaminio MJBF, LaCombe V, Kohn CW, et al. Common variable immuno-deficiency in a horse. J Am Vet Med Assoc. 2002; 9:1296-1302.
3. Flaminio MJBF, Tallbridge RL, Salles-Gomes COM, et al. Common variable immunodeficiency in horses is characterized by B cell depletion in primary and secondary lymphoid tissues. J Clin Immunol. 2009; 9:107-116.
4. Imai DM, Barr BC, Daft B, et al. Lyme neuroborreliosis in 2 horses. Vet Pathol. 2011; 48:1151-1157.
5. James FM, Engiles JB, Beech J. Meningitis, cranial neuritis, and radiculoneuritis associated with Borrelia burgdorferi infection in a horse. J Am Vet Med Assoc. 2010; 37:1180-1185.
6. Johnstone LK, Engiles JB. Retrospective evaluation of horses diagnosed with neuroborreliosis on postmortem examination: 16 cases (2004-2015). J Vet Intern Med. 2016; 30:1305-1312.
7. Kurmaran D, Eswaramoorthy S, et al. Crystal structure of outer surface protein C (OspC) from the lyme disease spirochete, Borrelia burgdorferi. EMBO J. 2001; 20(5):971-978.
8. Maglione PJ. Autoimmune and lymphoproliferative complications of common variable immunodeficiency. Curr Allergy Asthma Rep. 2016; 16:19.
9. Pandit C, Hsu P, van Asperen P, et al. Respiratory manifestations and management in children with common variable immunodeficiency. Paediatric Resp Rev. 2016; Epub ahead of print.
10. Pellegrini-Masini A, Bentz AI, Johns IC, et al. Common variable immuno-deficiency in three horses with presumptive bacterial meningitis. J Am Vet Med Assoc. 2005; 227:114-122.
11. Priest HL, Irby NL, Schlafer DH, et al. Diagnosis of Borrelia-associated uveitis in two horses. Vet Ophthalmol. 2012; 15:398-405.
12. Tallmadge RL, Shen L, Tseng CT, et al. Bone marrow transcriptome and epigenome profiles of equine common variable immunodeficiency patients unveil block of B lymphocyte differentiation. Clin Immunol. 2015; 160:261-276.
13. Tallmadge RL, Such KA, Miller KC, et al. Expression of essential B cell development genes in horses with common variable immunodeficiency. Mol Immunol. 2012; 51:169-176.
14. Tennet-Brown BS, Navas de Solis C, Foreman JH, et al. Common variable immunodeficiency in a horse with chronic peritonitis. Eq Vet Educ. 2010; 22:383-399.