Amyloid in the horse: a report of nine cases

Out of approximately 16,000 horses referred for clinical examination, nine had amyloidosis. Six of these horses had localised amyloid deposits in the wall of the nasal meatus and ventral turbinates associated with epistaxis. Horse 1 also developed malignant histiolymphocytic lymphosarcomas. The amyloid deposits were potassium permanganate-resistant and tryptophan-positive. Gel filtration of solubilised amyloid fibrils from Horse 1 revealed a major retarded fraction with an apparent molecular weight of 20 kD. This protein had an amino acid composition similar to human AL-amyloid proteins and horse immunoglobulin light chains. On Western blot a strong cross-reaction was observed between horse 1gG2a light chains and the Horse 1 amyloid. Horses 7 to 9 had suppurative verminous aneurysm, tuberculosis and an adrenal cortical adenoma, respectively, and had generalised amyloid deposits in liver and spleen. These amyloid deposits were found to be potassium permanganate-sensitive and positive for tryptophan. Gel filtration of solubilised amyloid fibrils from Horse 8 revealed a major retarded fraction (protein AA) with an apparent molecular weight of 10 kD. Immunoperoxidase-antiperoxidase staining showed the localised deposits to be negative or only weakly positive with antisera against bovine, hamster, dog and human protein AA and to be positive with anti-horse-one amyloid protein. The generalised deposits were found to be positive with the antisera against allogenic protein AA. The results of the potassium permanganate incubation, biochemistry, immunoblotting and immunochemistry, indicate that the localised amyloid of Horse 1 and most likely the amyloid of Horses 2 to 6, is of the AL-type. The generalised amyloid deposits were found to be of the AA type.     

Familial renal amyloidosis in Abyssinian cats

Medullary and glomerular amyloidosis, papillary necrosis, and secondary interstitial disease were diagnosed in eight related adult Abyssinian cats from two catteries. The lesions were similar to those in two unrelated mongrel cats with renal amyloidosis. Ultrastructurally, the patterns of amyloid deposition were as described in other species, although medullary deposition predominated. Potassium permanganate oxidation blocked Congo red staining of the deposits suggesting that they contained amyloid A protein (secondary amyloid). The disease may be a model of familial secondary amyloidosis and offers an opportunity to study the pathogenesis of both amyloid deposition and papillary necrosis. The histochemical characteristics of feline renal amyloid require careful attention to technique. Section thickness affects Congo red affinity and both dichroism as well as birefringence should be considered when interpreting staining reactions. Thioflavine-T may be the preferred stain for identification of small deposits of amyloid. Variation in section thickness markedly affected the degree of potassium permanganate oxidation.     

Systemic AL amyloidosis associated with multiple myeloma in a horse

AL amyloidosis is the most common type of systemic amyloidosis in humans, and it is frequently associated with multiple myeloma. But, AL amyloidosis is very rare in domestic animals. A 16-year-old Quarter horse gelding was diagnosed with systemic AL amyloidosis associated with multiple myeloma. Clinical problems were rapid weight loss, muscle atrophy, soft unformed stool, and ventral edema. Grossly, diffuse gastrointestinal hemorrhage, markedly thickened jejunal mucosa, and splenomegaly were present. Microscopically, diffuse severe amyloid deposits were present in the lamina propria of glandular stomach, duodenum, and jejunum. Much of the spleen and sternal bone marrow was replaced by neoplastic round cells, and multiple foci of amyloid were also present in the spleen and bone marrow. Electron microscopy revealed the neoplastic round cells to be of plasma cell origin, and the amyloid showed a strongly positive immunoreactivity with polyclonal anti-human immunoglobin lambda light-chain antisera. To our knowledge, this is the second report describing systemic AL amyloidosis in domestic animals-associated plasma cell neoplasia and the first associated with multiple myeloma, as is common in humans.     

The Pathogenesis of Reactive Systemic Amyloidosis

The diagnosis of amyloidosis is based on the presence of extracellular tissue deposits of proteinaceous material that demonstrate a characteristic green color when stained with Congo red and viewed under polarized light. Several different proteins are amyloidogenic but, in domestic animals, spontaneously occurring systemic amyloidosis is reactive in nature and characterized by the presence of amyloid protein AA. This type of systemic amyloidosis may occur secondary to chronic inflammatory or neoplastic disease, but in many instances no predisposing disease is found. A sustained increase in the serum concentration of serum amyloid A protein (SAA) is necessary but not sufficient for the development of reactive amyloidosis. Other inherited and acquired host-related factors are likely to be important in the development of reactive amyloidosis because this condition develops in few patients with chronic inflammatory disease. The tissue tropism of amyloid deposits varies with the amyloid protein itself and species affected. The consequences of amyloidosis for the host depend upon the tissues involved and the response of these tissues to the presence of the amyloid deposits. In domestic animals, reactive systemic amyloidosis is nephropathic, leading to end-stage renal disease, and the clinical presentation is that of uremia.     

Outcome of superficial keratectomy without conjunctival graft as a surgical technique for immune-mediated keratitis in horses: Four cases

This case report describes four horses with unilateral superficial or mid-stromal immune-mediated keratitis (IMMK) treated with a superficial keratectomy (SK) without a conjunctival graft. In two horses, the surgery was performed under general anaesthesia, and in two horses standing with sedation and local blocks. Results of this report show that SK is a viable treatment option in horses with chronic superficial and/or mid-stromal IMMK that can even be performed in the standing, sedated horse. When sufficient corneal tissue is removed, no recurrence is to be expected in the long-term follow-up (up to 31 months). In two horses, healing occurred without complications. Two horses developed a secondary bacterial infection post-operatively (Enterococcus faecalis and Staphylococcus aureus). In one case, this resulted in a pre-perforating melting corneal ulcer necessitating conjunctival pedicle graft surgery 13 days post keratectomy. In three horses, there was no recurrence of the IMMK with a long-term follow-up of 6–31 months. One case showed recurrence of IMMK in the cornea region surrounding the keratectomy 9 months after surgery.     

Immunohistochemical distribution of amyloid deposits in 25 cows diagnosed with systemic AA amyloidosis

The distribution of amyloid deposits was histopathologically and immunohistochemically examined in 25 cows aged 5 to 10 years that had been diagnosed with systemic AA amyloidosis. This examination revealed that amyloid deposits were also present in the hypophysis, ovary, uterus, mammary gland and skeletal muscle, in addition to the liver, kidney, spleen, pancreas, thyroid gland, adrenal gland, gastrointestinal mucosa, heart, lung and lymph nodes. The examined cows tended to have chronic inflammations, including chronic mastitis (six cases) or chronic pneumonia (four cases), which is thought of as a causative agent of AA amyloidosis. In contrast, five cases did not exhibit any chronic inflammation.     

Systemic amyloidosis in a mare

A mare with chronic cachexia had multiple skin nodules, abdominal masses (attached and free floating), and large lymph nodes. Fine-needle aspiration cytologic evaluation of a skin mass revealed multinucleated giant cells surrounding eosinophilic material. Histologic evaluation revealed extensive amyloid deposits within the masses, lymph nodes, and the interstitium of many organs. The presence of systemic (visceral) and organ-limited (cutaneous) forms of amyloid is rare in horses. Amyloid congophilia was retained after potassium permanganate oxidation. The fibrils were thus distinct from the AA (secondary) fibrils that are found in most cases of equine amyloidosis, suggesting that this mare may have had primary amyloidosis. Regardless of fibril type, the presence of multinucleated giant cells in association with eosinophilic material in cytologic preparations of skin nodules may suggest a differential diagnosis of amyloidosis in horses.     

Qualitative tear film and conjunctival goblet cell assessment of cats with corneal sequestra

OBJECTIVES: To evaluate the tear film qualitatively and conjunctival goblet cell numbers in cats with and without corneal sequestra. ANIMALS STUDIED AND PROCEDURES: This was a prospective evaluation of 11 cats with corneal sequestra and 14 control eyes that were either the contralateral normal eye when the sequestrum was unilateral or from control cats of similar age with no ocular disease. All cats in this study were examined by a veterinary ophthalmologist. The ophthalmic examinations included a neuro-ophthalmic evaluation, Schirmer tear tests, fluorescein staining, tear film break-up times, applanation tonometry, biomicroscopy, and indirect ophthalmoscopy. The palpebral conjunctiva at the dorsal nasal, ventral nasal, dorsal temporal and ventral temporal fornices were biopsied after topical anesthetic was applied to the cornea and conjunctiva. The conjunctival biopsies were fixed in formalin and sectioned routinely and stained with hematoxylin and eosin, and periodic acid-Schiff. These slides were examined by light microscopy by a blinded examiner. Goblet cell numbers were compared to conjunctival basal epithelial cell numbers by region. The goblet cell numbers by region from the eyes with sequestra was statistically compared to those from eyes without sequestra, with a student's paired t-test. Conjunctival swabs were collected from the cats with corneal sequestra and submitted for polymerase chain reaction for Herpes felis, Chlamydia psiitticia, and Mycoplasma felis. The corneal sequestra were removed by surgical keratectomy and fixed and stained routinely, and examined by light microscopy. RESULTS: No neurologic abnormalities were detected in any of the cats. The Schirmer tear tests (eyes with sequestra 14+/-5.1 mm/min; normal eyes 15+/-6.8 mm/min) and intraocular pressures (eyes with sequestra 21+/-6.6; normal eyes 22+/-5.8) were within normal reference ranges for cats. Biomicroscopic examinations revealed varied sizes and depths of brown- and amber-colored corneal sequestra. No abnormalities were noted on indirect ophthalmoscopic examinations. The tear film break-up time was 21 s (+/-12) for the normal eyes (n=14) and 14 s (+/-13) in eyes with corneal sequestra (n=11). The average goblet/epithelial cell ratios by region for the normal eyes and the eyes with sequestra respectively were 0.66, 0.56 for the dorsal nasal fornix, 0.68, 0.57 for the ventral nasal fornix, 0.63, 0.48 for the temporal dorsal fornix, and 0.55, 0.49 for the temporal ventral fornix. There were no significant differences in tear film break-up times and goblet cell numbers in eyes with corneal sequestra and those without sequestra. Three conjunctival swabs from two of 11 cats with sequestra were positive with PCR for Herpes felis virus. These included one cat with bilateral sequestra and one cat with unilateral corneal sequestrum. CONCLUSIONS: The pathogenesis of feline corneal sequestra does not appear to be linked primarily to abnormal goblet cell numbers, qualitative tear film abnormalities, and accelerated tear film break-up time.     

Pathology of AA amyloidosis in domestic sheep and goats

We describe the main pathologic changes in small ruminants affected by AA amyloidosis, together with the partial sequence of the protein involved. Twenty-one sheep and one goat were selected for presenting macroscopic kidney lesions compatible with systemic amyloidosis. Available tissue samples were studied by histologic, immunopathologic, and ultrastructural means. Renal lesions were characterized grossly by pale cortical surfaces with scattered, miliary, whitish-yellow foci and on cut cortical surfaces by straight, whitish-yellow striations. Gangrenous pneumonia was observed in 16 out of 21 affected sheep (76.2%), although other chronic inflammations were also observed. Amyloid was detected in all grossly affected kidneys using Congo red staining, lesions being most remarkable in glomeruli, affecting 95.5% of animals studied. Congophilic deposits were also observed in intertubular interstitium (68.2%) and medulla (57.1%). All amyloid-affected animals presented proximal convoluted tubule lesions, mostly characterized by an increase in diameter and by hyaline granular degeneration that were responsible for the macroscopic appearance of the kidney. Histologically, amyloid was also seen in blood vessels, spleen, liver, lymph nodes, gastrointestinal tract, and adrenal glands. All amyloid deposits demonstrated greenish-yellow birefringence with polarized light, and the antisera prepared against goat amyloid extracts specifically reacted with birefringent congophilic deposits of both sheep and goats. Ultrastructurally, these deposits were formed by masses of straight, nonbranching fibrils located predominantly in the basement membranes of glomerular capillaries and in the mesangium. Partial sequence of the protein in sheep and goats indicated a high degree of homology with the previously reported sequence of sheep Serum Amyloid A.     

Conjunctival malignant melanoma in a horse

A case of malignant melanoma originating from the conjunctiva of a horse is reported. The tumor exhibited locally aggressive behavior as evidenced clinically by recurrence following two treatment episodes including surgical excision on each occasion and one application of cryotherapy. The orbit was subsequently exenterated and histologically malignant conjunctival melanoma was confirmed. Histopathologic features included variable pigmentation with amelanotic sites demonstrating marked cellular and nuclear pleomorphism with high numbers of mitotic figures. Cords of neoplastic cells invaded the sclera and cornea. Following exenteration, the horse exhibited no recurrence of the tumor for five years before being lost to follow-up. To our knowledge, this is the first report of primary malignant conjunctival melanoma in a horse.     

Bilateral corneoconjunctival dermoids and nasal choristomas in a calf

A 5-day-old Angus x Hereford calf presented for bilateral haired masses on the eyes and nasolabial planum since birth. The masses were surgically excised from the inferonasal cornea and bulbar conjunctiva of both eyes and the left and right side of the dorsomedial nasolabial planum. Histopathology of the excised tissue confirmed bilateral corneoconjunctival dermoids with ectopic lacrimal glands, and bilateral nasal choristomas and ectopic nasal glandular tissue. Surgery was curative and healing was uneventful. Bilateral ocular dermoids in combination with nasal choristomas and ectopic glandular tissue have not been documented previously in cattle.     

Keratitis due to microfilariae in dogs: a newly recognized disease

Parasitic agents have been associated with keratitis, but a diagnosis of parasitic keratitis has not been commonly made in domestic animals. The purpose of this study was to describe the clinical and histopathological findings in seven dogs with chronic keratitis caused by microfilariae diagnosed in Brazil. All dogs presented with superficial corneal opacities of varying degrees affecting the perilimbal and central regions of the cornea, with other opaque areas appearing as crystalline deposits and corneal vascularization. The lesions were bilateral and were associated with mild‐to‐moderate conjunctival hyperemia. There was no history of blepharospasm or pruritus, and no subjects presented with epithelial erosions. Corneal biopsy revealed free microfilariae in the corneal stroma, with varying degrees of inflammation and collagen fiber destruction. The microfilariae were also found in skin lesions by skin snip technique. No adult worms were found in these dogs, and no dogs were on heartworm preventative before diagnosis. Monthly doses of oral ivermectin improved ocular and dermal lesions. One dog showed complete remission with the treatment. The species of the microfilariae was not identified.     

Expression of Toll-like receptors 2, 3, 4, 6, 9, and MD-2 in the normal equine cornea, limbus, and conjunctiva

Objective Human corneal cells have detectable levels of TLRs 1‐10. TLRs 2 and 4 are the major corneal receptors, recognizing the PAMPs associated with fungal invasion in humans. The conjunctiva and limbus contain TLRs 2, 4, and 9. Our purpose was to determine the expression of TLRs 2, 3, 4, 6, 9, and MD‐2 in the normal equine cornea, conjunctiva, and limbus. Methods Corneal, limbal, and conjunctival tissues were collected from seven euthanized horses having no evidence of ocular disease. RNA extraction with DNase‐1 digestion was performed followed by RT‐PCR to determine expression of TLRs 2, 3, 4, 6, 9, and MD‐2. Products were resolved by electrophoresis on 1.5% agarose gels and visualized using ethidium bromide staining. Results Expression of TLRs 2, 3, 4, 6, 9, and MD‐2 was present in the cornea, limbus, and conjunctiva of each horse, except one horse, where TLR3 expression was unable to be demonstrated in the dorsal and ventral conjunctiva. Conclusions Confirming the expression of TLRs in equine ocular tissues is an initial step in identifying how they play a role in infectious keratitis, particularly fungal. The results further support the use of equine ocular tissues as a model for human fungal keratitis. Studies of the TLR expression together with their cytokine profile induced during equine fungal keratitis may help further clarify the pathogenesis of the disease and possibly lead to the development of new treatment protocols for both equines and humans.     

Presence of opioid growth factor and its receptor in the normal dog,cat and horse cornea

Objective To determine if opioid growth factor (OGF, [Met⁵]enkephalin) and its specific receptor (OGFr) are present in normal cat, dog and horse cornea. Animals studied Normal dog, cat and horse. Procedure Corneas were obtained from animals euthanized for reasons unrelated to this project. One cornea from each of three normal cats, dogs and horses was evaluated. The right or left cornea from each animal was chosen randomly. Corneas were harvested and placed in corneal storage media for transport to The M.S. Hershey Medical Center of The Pennsylvania State University where immunocytochemistry techniques were used to demonstrate the presence and location of OGF and OGFr. Tissues were rinsed in Sorenson's phosphate buffer, immersed in 20% sucrose in buffer and then snap frozen in isopentane. Corneas were then embedded in OCT medium and 15 µm cryostat sections were created. Presence of OGF was determined by using a polyclonal antibody to [Met⁵]enkephalin and assessing immunoreactivity. OGFr presence was determined by using a previously characterized rabbit polyclonal antibody to the receptor. Results OGF and OGFr were identified in large quantities in the corneal epithelium of all three species. Conclusion Opioid growth factor and its specific receptor are present in the corneal epithelium of normal cats, dogs and horses. OGF is present in the cornea of many species and its presence is theorized to inhibit healing of injured tissue.     

Angiostatin and integrin alphavbeta3 in the feline, bovine, canine, equine, porcine and murine retina and cornea

PURPOSE: Angiogenesis is tightly controlled in the ocular tissues of domestic animals but its mechanisms are not fully understood. This is largely because of insufficient data on the expression of molecules that impact angiogenesis. Because angiostatin and one of its receptors integrin alphavbeta3 inhibit and promote angiogenesis, respectively, we hypothesized that the normal retina and cornea of domestic animals would express angiostatin but not integrin alphavbeta3. PROCEDURE: Normal eyes of the cat, cow, dog, horse, pig and rat were evaluated for angiostatin and integrin alphavbeta3 by light and electron immunocytochemistry and estern blots. RESULTS: Angiostatin was detected in the corneal epithelium of the cat, dog, horse, pig and rat, but was not found in cow corneal epithelium. Angiostatin was localized in the nerve fiber layer, ganglion cell layer, inner and outer plexiform layers, and the photoreceptor layer of the cat, cow, dog and rat. Horse and pig retinas showed additional staining in the matrix of the inner nuclear layer. Immunogold electron microscopy further confirmed angiostatin in cat retina. Western blots showed angiostatin in corneal and retinal homogenates. Integrin alphavbeta3 was absent in cornea and retina of all the species studied. CONCLUSION: These data show that angiostatin, an inhibitor of angiogenesis, is present while integrin alphavbeta3, which promotes angiogenesis, is absent in normal cornea and retina of the domestic animals in this study with the exception being angiostatin absence in cow corneal epithelium. Therefore, angiostatin may contribute to the anti-angiogenic environment in the normal domestic animal eye while its absence in the cow may contribute to greater propensity for corneal vascularization. Because integrin alphavbeta3 is one of the receptors for angiostatin, its absence may prevent angiostatin from killing normal retinal and corneal cells.     

Amniotic membrane transplantation for corneal surface reconstruction after excision of corneolimbal squamous cell carcinomas in nine horses

OBJECTIVE: The purpose of this study was to evaluate the usefulness and effectiveness of permanent amniotic membrane transplantation as an adjunctive treatment to superficial keratectomy alone or combined with strontium-90 irradiation for treatment of equine corneolimbal squamous cell carcinoma (SCC) to decrease corneal scarring and recurrence rate. STUDY: The retrospective case study included 11 horses (n = 12 eyes) diagnosed and treated for ocular SCC that involved the limbus and cornea. Nine of those horses (n = 9 eyes) were treated between 2002 and 2006, with superficial lamellar keratectomy alone or combined with strontium-90 irradiation and followed by placement of a permanent amniotic membrane graft in the surgical defect. The level of scarring (i.e. the clarity of the cornea) resulting with the use of amniotic membrane was subjectively compared to cases where a permanent bulbar conjunctival graft was performed following keratectomy combined with strontium-90 irradiation or cryotherapy (n = 3 eyes). Recurrence was defined as the postoperative and postirradiation regrowth of SCC in the same site and globe. RESULTS: The nine horses that received an amniotic membrane graft after keratectomy alone or combined with irradiation showed a minimal level of scarring in a cornea that regained a greater transparency in comparison to the horses that were treated with a bulbar conjunctival graft. All of the horses that received an amniotic membrane graft had 226 +/- 218 days of follow-up without tumor recurrence (mean +/- SD), ranging from 21 days to 778 days. CONCLUSIONS: The combination of superficial keratectomy alone or associated with beta-irradiation and permanent amniotic membrane transplantation is an effective treatment of corneal or corneolimbal SCC in horses. The placement of an amniotic membrane material represents an alternative surgical procedure to bulbar conjunctival grafts, especially if there is a lack of bulbar conjunctiva tissue available after tumor resection or if a particularly large corneal resection is necessary. The amniotic membrane is incorporated into the corneal defect and seems to create noticeably much less scarring than a corneal defect covered by bulbar conjunctiva.     

Episcleral cyclosporine A implants for the management of unilateral keratoconjunctivitis sicca in an 8‐year‐old mare

An 8‐year‐old mare was presented for investigation of a 1‐month history of blepharospasm, eyelid swelling, corneal edema, and ocular discharge of the right eye (OD). Ophthalmic examination confirmed mucopurulent ocular discharge, conjunctival hyperemia, and a dry, dull appearance to the cornea OD. Schirmer tear test results confirmed an absence of tear production OD (0 mm/min) consistent with keratoconjunctivitis sicca. Treatment with topical 0.2% cyclosporine A resulted in an improvement in clinical signs. An episcleral cyclosporine A implant was placed under standing sedation 5 days after initial presentation. Re‐examination 9 days post‐operatively confirmed that the mare's tear production in the right eye had improved and no further clinical signs had been observed. Topical medications were gradually discontinued. Re‐examinations performed up to 12 months postsurgery showed no recurrence of clinical signs and no adverse effects of the implant. To our knowledge, this is the first report of the use of a cyclosporine A implant in the management of KCS in a horse and highlights its potential as an effective, alternative therapy in the management of KCS in horses.     

Presumed primary ocular lymphangiosarcoma with metastasis in a miniature horse

A 7‐year‐old, 153.0‐kg American Miniature mare presented for evaluation of keratoconjunctivitis of the right eye (OD). A superior palpebral conjunctival mass and stromal keratitis were diagnosed. The incisional biopsy diagnosis was a presumptive corneal hemangiosarcoma. Transpalpebral enucleation was performed, and histopathologic evaluation confirmed angiosarcoma of the conjunctiva, cornea, and extraocular muscles. The horse developed progressive epistaxis and orbital swelling following surgery. A systemic workup was performed 3 months after enucleation, revealing regrowth within the orbit and marked cranial cervical lymphomegaly, suggestive of metastasis. Humane euthanasia was performed, and necropsy confirmed a locally invasive periorbital tumor with metastasis to the submandibular tissue, submandibular lymph node, and thoracic inlet. Histopathologic evaluation of necropsy specimens revealed polygonal to spindle neoplastic cells lining neoplastic vascular channels lacking erythrocytes. Immunohistochemically, the neoplastic cells labeled strongly positive for PROX‐1, vimentin, CD‐31, VEGF, weakly positive for factor VIII‐related antigen, and negative for collagen IV. Based on the clinical, histological, and immunohistochemical features of this tumor, a primary ocular lymphangiosarcoma with metastasis was diagnosed.     

Glycohistochemical characterization of histologically normal nasal mucosa and enzootic nasal tumor of sheep

Objective-To determine glycohistochemical characteristics of enzootic nasal tumors (ENTs) of sheep, compare results for ENT with those of histologically normal nasal mucosa of sheep, and identify the histologic origin of ENT. Sample-ENT and nasal mucosa samples obtained from cadavers of 5 adult Lacaune sheep with ENT and 5 Lacaune sheep unaffected by ENT, respectively. Procedures-Samples of ENT and nasal mucosa were collected from cadavers of sheep and sectioned. Conventional and lectin histochemical analyses were used to identify glycoconjugates in tissue sections on the basis of their principal chemical groups and principal terminal or internal oligosaccharidic glucidic residues, respectively. Results-ENTs had papillary and tubular portions. Cells in the papillary portion of ENTs had secretion and surface glycoconjugates, which included sulfated glycosaminoglycans and neutral and sialilated glycoproteins. Cells in the tubular portion of ENTs had surface glycoconjugates, which included neutral and sialilated glycoproteins. Both portions of ENTs had C(4)-acetylated sialoderivatives that were not detected in sections of histologically normal nasal mucosa. Conclusions and Clinical Relevance-The papillary portion of ENTs in sheep may originate from respiratory glands and goblet cells. The tubular portion of ENTs in sheep may originate from olfactory glands. Presence of C(4)-acetylated sialoderivatives in cells of ENTs could confer resistance against pathogens to those cells.